Oral delivery of angiotensin converting enzyme 2 (ACE2) or angiotensin-(1-7) bioencapsulated in plant cells attenuates pulmonary hypertension, cardiac dysfunction and development of autoimmune and experimental induced ocular disorders

ABSTRACT

Experiments have also been performed which indicate that this approach is also suitable for the treatment or inhibition of experimental uveitis and autoimmune uveoretinitis These studies provide proof-of-concept for a novel low-cost oral ACE2 or Ang-(1-7) delivery system using transplastomic technology for pulmonary and ocular disease therapeutics.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of PCT/US2014/061428 filed Oct. 20, 2014 which in turn claims priority to US Provisional Application Nos. 61/892,717, 61/943,754 and 61/952,078 filed Oct. 18, 2013, Feb. 24, 2014 and Mar. 12, 2014 respectively. Each of these applications is incorporated herein by reference as though set forth in full.

STATEMENT OF FEDERAL FUNDING

This invention was made with government support under grant nos. HL099980, HL102033, HL106687, HL109442, EY021752 and EY21721 awarded by the National Institutes of Health. The U.S. Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Numerous publications and patent documents, including both published applications and issued patents, are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

Pulmonary hypertension (PH) is a devastating lung disease characterized by elevated blood pressure in the pulmonary circulation, which eventually leads to right-heart failure and death.¹ Although significant advances have been made in recent years to improve the quality of life of patients with PH; none of the current treatments are successful in reversing PH or decreasing mortality. This has led to the realization that novel mechanism based therapies must be developed to accomplish this goal.²

It is well-recognized that activation of the vasodeleterious axis of the renin angiotensin system (RAS), comprising of angiotensin-converting enzyme (ACE), angiotensin II (AngII) and angiotensin type I receptor (AT1R) is involved in the development of PH.^(3,4) However, the clinical use of ACE inhibitors or AT1R blockers have yielded mixed results, thereby failing to reach a consensus opinion regarding their use for PH therapy. Nonetheless, the recent discovery of a close homolog of ACE, angiotensin converting enzyme2 (ACE2) has resulted in the re-evaluation of the role of RAS in PH.^(5,6) ACE2 is widely expressed in the lungs,⁷ predominantly on the pulmonary vascular endothelium, and catalyzes the conversion of AngII to Angiotensin-(1-7) [Ang-(1-7)]. Ang-(1-7) is a vasoactive heptapeptide that mediates its effects by stimulating the Mas receptor.⁸ Thus, ACE2-Ang-(1-7)-Mas receptor constitutes the vasoprotective axis of RAS, which counterbalances the deleterious actions of the ACE-AngII-AT1R axis.

Recent reports indicate that decreased tissue and circulating levels of ACE2 are associated with lung diseases in humans.^(9,10) On the other hand, restoration of ACE2 through genetic overexpression, administration of recombinant protein or use of pharmacological ACE2 activators resulted in cardiopulmonary protective effects against animal models of pulmonary diseases.^(11,15) These findings provided compelling evidence for initiating clinical trials with recombinant ACE2 or Ang-(1-7) in treating pulmonary disorders. Although clinical trials are currently underway (ClinicalTrials.gov; NCT01884051), the cost of manufacturing, protein stability, repetitive intravenous dosing and patient compliance pose major impediments in realizing full therapeutic potential of this therapy.

The renin-angiotensin system (RAS) plays an important role not only in the cardiovascular homeostasis, but also in the pathogenesis of inflammation and autoimmune dysfunction in which Angiotensin II (Ang II) functions as the potent proinflammatory effector via Angiotensin Type 1 receptor (AT1 receptor). Most components of RAS have been identified in every organ including the eye. The tissue-specific RAS is believed to exert diverse physiological effects locally independent of circulating Ang II (Paul et al., (2006) Physiol Rev. 86:747-803). Several studies have shown that ACE2/Ang-(1-7)/Mas axis also influences inflammatory responses and negatively modulates leukocyte migration, cytokine expression and release, and fibrogenic pathways (Qui et al. (2014) Invest. OPthalmol Vis Sci. 55:3809-3818) We have recently shown that increased expression of ACE2 and Ang-(1-7) reduced diabetes-induced retinopathy and inflammation in both mouse and rat models of diabetic retinopathy (Rawas-Qalaji et al., (2012) Curr Eye Res. 37:345-356), activation of endogenous ACE2 activity reduced endotoxin-induced uveitis (Kwon et al., (2013) Adv. Drug Deliv Rev 65:782-799), providing the proof-of-concept that enhancing the protective axis of RAS is a promising therapeutic strategy for ocular inflammatory diseases.

However, the ability to deliver drugs efficiently to the retina or the brain remains a key challenge due to anatomic barriers and physiological clearance mechanisms [13].

SUMMARY OF INVENTION

In accordance with the present invention a composition comprising lyophilized plant material comprising a therapeutic protein produced in chloroplasts which retains biological function in lyophilized form is provided. Surprisingly oral administration of said material to a patient in need thereof is effective to produce a beneficial therapeutic result. In one embodiment of the invention, the plant material comprises leaves obtained from a homoplasmic plant, and the therapeutic protein is a fusion protein comprising angiotensin-converting enzyme 2 (ACE-2), and cholera non toxic B subunit (CTB) and exerts beneficial cardioprotective effects. In another embodiment, the plant material comprises leaves obtained from a homoplasmic plant, and the therapeutic protein is a fusion protein comprising angiotensin-(1-7) (Ang-(1-7)), and cholera non toxic B subunit (CTB), and provides a cardioprotective effect. The plant species for transgenic expression of said therapeutic protein can include, without limitation, lettuce, carrots, cauliflower, cabbage, grass, low-nicotine tobacco, spinach, kale, and cilantro. The fusion proteins described above can contain a hinge peptide and furin cleavage site between said CTB and said ACE-2 or said Ang1(1-7).

In a particularly preferred embodiment, the lyophilized plant material comprises a combination of ACE-2 and angiotensin-(1-7) (SEQ ID NO: 29). In another aspect of this embodiment, the ACE2 sequence is codon optimized and is encoded by SEQ ID NO: 24.

In another aspect, a method for the treatment of pulmonary hypertension comprising administration of an effective amount of the compositions described above to a patient in need thereof is the disclosed wherein the composition exerts a cardioprotective effect comprising at least one of improved right heart function, decreased pulmonary vessel wall thickness, and a lack of altered basal system blood pressure, said method optionally comprising assessing said effects after administration of said composition. In certain embodiments, the patient is assessed for cardioprotective effects.

In another aspect of the invention, the compositions described above can be used to advantage to reduce ocular inflammation. Thus, the present invention also provides a method for treating or delaying the onset of uveoretinitis in a subject in need thereof comprising oral administration of a therapeutically effective amount of the compositions described above, wherein the administration is effect to reduce ocular inflammation in said subject, the method optionally comprising assessing said reduction in ocular inflammation in said subject. In certain embodiments, the subject is assessed for a reduction in ocular inflammation.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1G. Characterization, concentration and evaluation of pentameric structure of CTB-ACE2 and CTB-Ang-(1-7) expressed in plant chloroplasts. (FIG. 1A) Schematic representation of CTB-ACE2 and CTB-Ang-(1-7) gene cassettes and flanking regions. Southern blot analysis of (FIG. 1B) CTB-ACE2 and (FIG. 1C) CTB-Ang-(1-7) transplastomic lines. HindIII-digested untransformed (UT) and transformed (lane 1, 2, and 3) genomic DNA was probed with P³²-labeled flanking sequence. Quantification of (FIG. 1D) CTB-ACE2 and (FIG. 1E) CTB-Ang-(1-7) as a percentage of the total leaf proteins (TLP). (FIG. 1F) GM1 binding assay of CTB-ACE2 and CTB-Ang-(1-7). CTB, non-toxic cholera B subunit (1 ng); UT, untransformed wild type; F, fresh; L, lyophilized; BSA, bovine serum albumin (1%, w/v). (FIG. 1G) Western blot analysis of CTB-Ang-(1-7) in non-reducing condition without boiling and DTT. Lanes 1, 2, and 3: 10, 15, and 20 ng of CTB; total homogenate of CTB-Ang-(1-7): 0.2, 0.4, 0.8, and 1.6 μg. The pentameric structures for the CTB alone and the fusion protein are indicated by arrow head and arrow, respectively. Data shown are mean±SD of three independent experiments.

FIGS. 2A-2H. Oral administration of bioencapsulated ACE2 or Ang-(1-7) prevents MCT-induced PH. (FIG. 2A) Measurement of right ventricular systolic pressure (RVSP) in normal controls and MCT-challenged rats that were either untreated or orally fed with wild type leaf material or gavaged with bioencapsulated ACE2/Ang-(1-7). (FIG. 2B) Right ventricle (RV) hypertrophy, measured as the ratio of RV to left ventricle (LV) plus interventricular septum (S) weights [RV/(LV+S)]. Measurement of (FIG. 2C) right ventricular end-diastolic pressure (RVEDP), (FIG. 2D) +dP/dt, and (FIG. 2E) −dP/dt. Echocardiography data representing (FIG. 2F) ejection fraction (EF), (FIG. 2G) ratio of the right to left end diastolic area, signifying right heart dilation and (FIG. 2H) the blood flow rate in the right ventricular outflow tract (RVOT). Data shown are mean±SEM. ***P<0.001 vs. control rats and #P<0.05 vs. untreated or wild type leaf fed MCT-rats. n=6-8 animals/group.

FIGS. 3A-3D. Oral feeding of bioencapsulated ACE2 or Ang-(1-7) improves cardiac function in PH as measured by echocardiography. Echocardiography was performed at the end of the study in M mode at the parasternal short axis view at the papillary muscle level as described in the Methods. Ejection fraction of the left ventricle was calculated using the following formula: (End Diastolic Volume−End Systolic Volume/End Diastolic Volume)×100. In the parasternal short axis view, the transducer was slightly angled to record the image of both right and left ventricle and this image was used to analyze the right and left ventricular end diastolic area. (FIG. 3A) End Diastolic Area of Left Ventricle (LV EDA), as visualized by echocardiography. LV EDA was significantly reduced in PH animals and the oral feeding of ACE2 or Ang-(1-7) prevented the reduction in the LV EDA. (FIG. 3B) End Diastolic Area of Right Ventricle (RV EDA), as visualized by echocardiography. RV EDA was significantly increased in the PH animals demonstrating the right ventricular dilation. Oral feeding of ACE2 or Ang-(1-7) significantly reduced the RV dilation and improved the cardiac function. (FIG. 3C) Ejection Fraction of Left Ventricle, as visualized by echocardiography. LV EF was slightly reduced in PH animals. Data represents mean±SEM with n=5 animals. (FIG. 3D) Ejection fraction of Right Ventricle, as visualized by echocardiography. RV EF was significantly reduced in the PH animals demonstrating the cardiac dysfunction. Oral feeding of ACE2 or Ang-(1-7) attenuated the MCT induced decrease in RV ejection function. (MCT+ACE2-P and MCT+Ang-(1-7)-P represents the prevention protocol, while MCT+ACE2-R and MCT+Ang-(1-7)-R represents the reversal protocol. MCT+C-500 and MCT+C-250 represent the combination treatment using 500 mg and 250 mg respectively in the reversal protocol.) In, both figures, data represent mean±SEM with n=5 animals *** denoting p<0.001 as compared with normal controls, while # representing p<0.05 Vs untreated and wild type plant material fed MCT animals as assessed by One-Way ANOVA followed by Newman-Keuls test.

FIG. 4. Oral feeding of bioencapsulated ACE2 or Ang-(1-7) increases circulating levels of Ang-(1-7) in both prevention and reversal protocols. (A) Data indicates significant elevation in circulating levels of Ang-(1-7) in both prevention and reversal protocols (n=5 rats per group). (MCT+ACE2 (P) and MCT+Ang-(1-7) (P) represents the prevention protocol, while MCT+ACE2 (R) and MCT+Ang-(1-7) (R) represents the reversal protocol. Data represent mean±SEM with n=5 animals ** denoting p<0.01 as compared with normal controls, untreated and wild type plant material fed MCT animals as assessed by One-Way ANOVA followed by Newman-Keuls test.

FIGS. 5A-5D. Oral feeding of bioencapsulated ACE2 or Ang-(1-7) exerts cardioprotective effects. (FIG. 5A) Data indicates significant elevation of right ventricular systolic pressure (RVSP) after 2-weeks of MCT administration, signifying induction of PH (n=5 rats per group). Data are expressed as mean±SEM; * p<0.05 versus controls using student t-test. Measurement of (FIG. 5B) right ventricular end-diastolic pressure (RVEDP), (FIG. 5C) +dP/dt, and (FIG. 5D) −dP/dt in normal controls and MCT-challenged rats that were either untreated or orally fed with wild type leaf material or gavaged with bioencapsulated ACE2/Ang-(1-7). Data are expressed as mean±SEM; ***p<0.001; versus controls and #p<0.05 versus MCT group. n=5 animals per experimental group.

FIGS. 6A-6C. Oral feeding of bioencapsulated ACE2 or Ang-(1-7) exerts anti-fibrotic and anti-remodeling effects in the prevention protocol. (FIG. 6A) Interstitial collagen deposition in the right ventricle. (FIG. 6B) Staining for α-smooth muscle actin to quantify medial wall thickness of the pulmonary arteries measuring less than 50 μm. Scale bar denotes 10 μm (FIG. 6C) ACE2 activity was measured in rat sera (10 μl) collected from different experimental groups AFU: Arbitrary fluorescence units. Data represents mean±SEM with * denoting p<0.05 Vs other groups, ** denoting p<0.01 as compared with controls, while # representing p<0.05 vs. untreated and wild type plant material fed MCT-rats as assessed by One-Way ANOVA followed by Newman-Keuls test.

FIGS. 7A-7G. Oral treatment with ACE2 or Ang-(1-7) arrests disease progression and attenuates cardiopulmonary remodeling. (FIG. 7A) Individual values of the right ventricle systolic pressure (RVSP) from different experimental groups of the reversal protocol. (FIG. 7B) RV/(LV+S) values from individual animals, denoting right heart hypertrophy. Echocardiography data representing (FIG. 7C) ratio of the right to left ventricle end diastolic area, (FIG. 7D) ejection fraction (EF), and (FIG. 7E) the blood flow rate in the right ventricular outflow tract (RVOT) of the different experimental groups. (FIG. 7F) Representative photographs and quantification of interstitial fibrosis (FIG. 7G) Measurement of vessel wall thickness following α-smooth muscle actin staining of the pulmonary arteries (<50 μm). Scale bar denotes 10 μm. Data shown are mean±SEM. ** P<0.01, ***P<0.001 vs. control rats and #P<0.05 vs. untreated or wild type leaf fed MCT-rats. n=6-8 animals/group.

FIG. 8A-8H. Combination therapy with ACE2 and Ang-(1-7) rescues established PH. (FIG. 8A) Measurement of right ventricular systolic pressure (RVSP) in MCT rats treated with a combination of either 500 mg or 250 mg each of ACE2 and Ang-(1-7). (FIG. 8B) Data representing right ventricular hypertrophy as a ratio of RV/(LV+S). Measurement of (FIG. 8C) right ventricular end-diastolic pressure (RVEDP), (FIG. 8D) +dP/dt, and (FIG. 8E) −dP/dt from the combination study. Echocardiography data representing (FIG. 8F) ejection fraction (EF), (FIG. 8G) ratio of the right to left end diastolic area and (FIG. 8H) the blood flow rate in the right ventricular outflow tract (RVOT). Data shown are mean±SEM. ***P<0.001 vs. control rats and #P<0.05 vs. untreated or wild type leaf fed MCT-rats. n=6-8 animals/group.

FIGS. 9A-9B. Combination of ACE2 and Ang-(1-7) decreases ventricular fibrosis and attenuates pulmonary vascular remodeling. (FIG. 9A) Representative photographs of collagen staining and quantitative analysis of right ventricular fibrosis following 2-week treatment with combination therapy. (FIG. 9B) Measurement of vessel wall thickness of the pulmonary arteries (<50 μm). Scale bar denotes 10 μm. Data are expressed as mean±SEM; **p<0.01; versus controls and #p<0.05 versus untreated and wild type plant material fed MCT-rats. n=5-7 animals per experimental group.

FIGS. 10A-10J. Effects of ACE2 or Ang-(1-7) treatment on the lung renin-angiotensin system (RAS), pro-inflammatory cytokines and autophagy (prevention protocol). Relative change in lung mRNA levels of (FIG. 10A) Angiotensin-converting enzyme (ACE), (FIG. 10B) angiotensin-converting enzyme 2 (ACE2), (FIG. 10C) ACE/ACE2 ratio, (FIG. 10D) AT1R, (FIG. 10E) AT2R and (FIG. 10F) AT1R/AT2R receptor. Relative mRNA levels of lung pro-inflammatory cytokines, (FIG. 10G) tumor necrosis factor (TNF)-α, (FIG. 10H) transforming growth factor (TGF)-β and (FIG. 10I) toll-like receptor-4 (TLR-4) from the MCT study. Autophagy marker, LC3-II is increased in the lungs of MCT-exposed animals. (FIG. 10J) Immunoblot and densitometry analysis of the lung LC3I/II protein expression. Data are expressed as mean±SEM. * P<0.05, ** P<0.01, and *** P<0.001 versus control rats. #P<0.05 versus MCT group.

FIGS. 11A-11J. Effects of Monotherapy as well as the combination therapy on the lung RAS components, pro-inflammatory cytokines and autophagy in the reversal protocol. Data represent relative changes in lung mRNA levels of (FIG. 11A) Angiotensin-converting enzyme (ACE), (FIG. 11B) angiotensin-converting enzyme 2 (ACE2), (FIG. 11C) ACE/ACE2 ratio, (FIG. 11D) Angiotensin type 1 receptor (AT1R), (FIG. 11E) Angiotensin type 2 receptor (AT2R) and (FIG. 11F) AT1R/AT2R ratio. Relative mRNA levels of lung pro-inflammatory cytokines, (FIG. 11G) tumor necrosis factor (TNF)-α, (FIG. 11H) transforming growth factor (TGF)-β and (FIG. 11I) toll-like receptor-4 (TLR-4) from the same study. (FIG. 11J) Immunoblot and densitometry quantification showing lung LC3I/II protein expression. Data are expressed as mean±SEM. * p<0.05 and ** p<0.01 versus control rats, while #P<0.05 versus MCT group.

FIGS. 12A-12E. Evaluation of proper formation of pentameric structure and lyophilization for the CTB fusion proteins. FIG. 12A. Western blot analysis to investigate the proper folding and assembly of CTB-Ang-(1-7) expressed in chloroplasts. Four micrograms of total leaf protein were loaded for each lane with (+) or without (−) treatment of denaturing agents. CTB, purified non-toxic cholera B subunit (10 ng); UT, untransformed wild type; DTT, 100 mM; boiling, incubation of samples in boiling water for 3 min; H, homogenate total leaf protein; S, supernatant fraction after centrifugation of the total leaf protein; Arrows and numbers, locations of monomer and oligomers of CTB-Ang-(1-7); P, pentamer-pentamer complexes. FIG. 12B. Western blot analysis for the comparison of the level of CTB-Ang-(1-7) in lyophilized (L) and fresh (F) leaves. Equal amount of lyophilized and fresh leaf material (10 mg) was extracted in same volume (300 μl ) of extraction buffer. 1× represents 1 μl of homogenate protein resuspended in extraction buffer. The samples were boiled in DTT prior to loading on SDS acrylamide gel. Purified CTB standard protein was loaded as indicated for densitometric analysis. FIG. 12C. and FIG. 12D. Comparison of the level of CTB-Ang-(1-7) and −ACE2 in lyophilized (L) and fresh (F) leaves. Data are means±SD of three independent experiments. FIG. 12E. GM1 binding assay of CTB-ACE2 and −Ang-(1-7). Extracted total protein samples were serially diluted up to 10 pg/ul, which means 11 on the Y axis, and used for GM1 binding assay. The binding affinity was read at 450 nm then an absorbance of ≥0.1 after background signal substraction was determined as positive. Two and three different batches were examined for fresh and lyophilized leaf materials, respectively, and indicated as black diamond. CTB, purified non-toxic cholera B subunit (black circle); UT, untransformed wild type (black triangle); F, fresh; L, lyophilized; 12 and 15, 12- and 15-month storage at room temperature. *p<0.001 (versus fresh); #p<0.001 (versus WT).

FIGS. 13A-13D. ACE2 activity assay using protein samples extracted from CTB-ACE2 transplastomic and untransformed leaf materials (WT). FIG. 13A. Assay buffer containing the substrate was also used as a negative control. FIG. 13B. Increased ACE2 in both serum and retina in mice fed with CTB-ACE2 leaf material detected by Western blotting using an anti-ACE2 polyclonal antibody. FIG. 13C. ACE2 activities in serum and retina from mice fed with either fresh (F, 500 mg/mouse), or lyophilized (L, 50 mg/mouse) CTB-ACE2 leaf materials, compared to mice fed with wild type (WT) leaf materials; (n=5 per group). The experiment was repeated at least twice with similar results. * p<0.05 (versus WT leaf). FIG. 13D. A graph showing results from an ELISA assay demonstrating the presence of Ang-(1-7) in plasma and retina after oral administration.

FIGS. 14A-14C. Histological evaluation of EIU mice. The mice were orally administered with Wild-type, CTB-ACE2 and CTB-Ang-(1-7) expressed leaf material for five days before LPS (25 ng/eye) injection. Eyes were enucleated 24 hr after LPS injection, fixed and processed for paraffin sections and stained with H&E. FIG. 14A. Representative photographs of the iris ciliary body, anterior chamber and posterior chamber. Original magnifications 20×. Bar=50 μm. FIG. 14B. Histopathologic score evaluation. Inflammatory cells per section in the iris ciliary body, anterior chamber and posterior chamber were counted from H&E stained paraffin sections from eyes at 24 h after EIU induction. Values on y-axis represent no. of infiltrating inflammatory cells/section. Results are given as mean+SD; (n=6 per group); *P<0.05 (versus WT+LPS group). FIG. 14C. Histological evaluation of EIU mice. The mice were orally administered with different doses of lyophilized plant cells expressing CTB-ACE2 for four days before LPS (25 ng/eye) injection. Eyes were enucleated 24 hr after LPS injection, fixed and processed for sections and stained with H&E and Histopathologic score was evaluated by at least two individuals. Inflammatory cells per section in the iris ciliary body, anterior chamber and posterior chamber were counted from H&E stained sections from eyes at 24 h after EIU induction. Values on y-axis represent no. of infiltrating inflammatory cells/section. Results are given as mean+SD; (n=6 per group); *P<0.05 (versus WT+LPS group).

FIGS. 15A-15B. Real-time reverse transcriptase (RT)-PCR analysis of ocular mRNA levels of inflammatory cytokines (FIG. 15A) and RAS genes (FIG. 15B). Values on y-axis represent fold difference compared to age-matched wild-type control ocular samples for each gene. WT ctrl, non-fed wild-type control; WT+LPS, WT leaf fed & LPS injected; ACE2+LPS, CTB-ACE2 expressed leaf fed & LPS injected; Ang-(1-7)+LPS, CTB-Ang-(1-7) expressed leaf fed & LPS injected. Data expressed as mean+SD; (n=4 per group); * P<0.05 (versus WT+LPS group).

FIGS. 16A-16G. Clinical evaluation of EAU from fundoscopic photographs. EAU was induced in B10.RIII mice by immunization with IRBP in CFA. The fundoscopic images were obtained on day 14 after immunization. Representative fundus image from WT leaf fed mice (FIG. 16a , FIG. 16b ); CTB-ACE2 expressed leaf fed mice (FIG. 16c , FIG. 16d ); and CTB-Ang-(1-7) expressed leaf fed mice (FIG. 16e , FIG. 16f ). FIG. 16G. Clinical EAU scores. Clinical EAU score was evaluated on a scale of 0-4. Values on y-axis represent the average of clinical scores given on fundus images. Results are given as mean+SD; (n=5 per group); *P<0.05 (versus WT fed group).

FIGS. 17A-17B. Assessment of retinal thickness on OCT images from EAU mice. Horizontal and cross sectional OCT images were obtained on day 14 after immunization. The retinal thickness was measured and averaged from five different frames of horizontal OCT scan images of single eye. FIG. 17A. Representative fundus projection (left panel) and B-scan (right panel) images from WT leaf fed mice; CTB-ACE2 expressed leaf fed mice; and CTB-Ang-(1-7) expressed leaf fed mice. FIG. 17B. Retinal thickness measured from OCT images. Values on y-axis represent the average of retinal thickness calculated manually from B-scan OCT images. Results are given as mean+SD; (n=5 per group); *P<0.05 (versus WT fed group).

FIGS. 18A-18C. Histological evaluation of EAU. H&E staining, magnifications 4×, Bar=200 μm; and 20×, Bar=50 μm. FIG. 18A. Representative micrographs from animals fed with WT leaf fed mice, CTB-ACE2, and CTB-Ang-(1-7) leaf materials. Images of histological analysis show severe retinal folding, loss of the photoreceptor layer and massive inflammatory cell inflammation in the vitreous, retina and subretinal space in WT leaf fed group; moderate to minimum infiltration, photoreceptor damage, retinal folding was observed in CTB-ACE2 expressed leaf fed group; a minor infiltration of cells and retinal folding was observed in the CTB-Ang-(1-7) leaf fed group. FIG. 18B. Histopathology scores. CTB-ACE2 and CTB-Ang-(1-7) leaf fed groups showed a reduced EAU histological grade compared to controls fed with WT leaf. FIG. 18C. Evaluation of infiltrating inflammatory cells in the posterior chamber. Inflammatory cells/section in the posterior chamber were counted on 14th day after EAU induction. Values on y-axis represent no. of infiltrating inflammatory cells/section. Results are given as mean+SD; (n=5 per group); *P<0.05 (versus WT leaf fed group).

FIGS. 19A-19C. Evaluation of EAU from fundoscopic photographs, OCT and histopatholgy. EAU was induced in B10.RIII mice by immunization with IRBP in CFA. The treatment with CTB-Ang-(1-7) was delayed. The fundoscopic images were obtained on day 14 after immunization. FIG. 19A. Clinical EAU scores. Clinical EAU score was evaluated on a scale of 0-4. FIG. 19B. Retinal thickness measured from OCT images. Horizontal and cross sectional OCT images were obtained on day 14 after immunization. The retinal thickness was measured and averaged from five different frames of horizontal OCT scan images of single eye. FIG. 19C. Histopathology scores. CTB-Ang-(1-7) leaf fed groups showed a reduced EAU histological grade compared to controls fed with WT leaf. Values on y-axis represent the average of clinical scores given on fundus images. Results are given as mean+SD; (n=6 per group); *P<0.05 (versus WT fed group).

FIGS. 20A-20D. Codon distribution of psbA (FIG. 20A), chloroplast based codon tables, native and codon-optimized (N and O) ACE2 gene. Codon-optimized ACE2 were optimized by changing the rare codons and codons usage frequency to resemble the chloroplast psbA gene (FIGS. 20B, C and D). Sequence alignments of native and codon-optimized (N and O) ACE2 gene. Any different nucleotides of codon-optimized sequences are marked in light grey. Nat: native sequence (SEQ ID NO: 25); CO-N: codon-optimized sequence obtained from new version of optimizer (SEQ ID NO: 26); CO-0: codon-optimized sequence obtained from old version of optimizer (SEQ ID NO: 27). The corresponding amino acid sequence (SEQ ID NO: 28) is shown below the aligned nucleotide sequences.

FIGS. 21A and 21B. Native and Optimized CTB-ACE2 Constructs and Sequences. FIG. 21A. Native sequence (SEQ ID NO: 23). FIG. 21B. Codon optimized sequence (SEQ ID NO: 24).

FIGS. 22A-22D. Selection and regeneration of CTB-ACE2 transplastomic lettuce. (FIG. 22A) transplastomic lettuce shoots undergoing first round of selection; (FIG. 22B) second rounds of selection; CTB-ACE2 lettuce acclimated in growth chamber (FIG. 22C) and grown in greenhouse (FIG. 22D).

FIG. 23. Western blot analysis of ACE2-expressed transplastomic lettuce plants. 5 total protein extracted from wild type (WT), native CTB-ACE2 expressed (N) and codon-optimized CTB-ACE2 expressed (CO) were detected by anti-CTB antibody. CTB (5 ng) was loaded as positive control. The homoplasmic lettuce plants expressing codon-optimized ACE2 showed 7.7-fold higher expression than the native ACE2.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments disclosed herein provide a method for treating subjects suffering from chronic diseases such as cardiovascular disease, cardiopulmonary disease, and other lung diseases involving pulmonary fibrosis, diabetes-related micro- and macro-vascular diseases, metabolic syndrome, stress-related disorders and ocular disorders. For example, studies have shown that overexpression of ACE2 or Ang-(1-7) in the lungs or its activation by small molecule activators prevents pulmonary hypertension-induced lung pathophysiology (Shenoy et al, Curr Opin Pharmacol 2011, 11:150-5. Shenoy et al, Am J Respir Crit Care Med. 2013, 187(6):648-57). In addition, ACE2 activators attenuate ischemia-induced cardiac pathophysiology (Qi et al, Hypertension. 2013, 62(4):746-52) and produce beneficial effects on dysfunctional diabetic EPCs (Jarajapu et al, Diabetes, 2013, 62, 1258-69. Since this enzyme is a protein, the only effective way to increase its levels in a diseased state is to administer it intravenously or intramuscularly. Both of these methods of delivery are extremely inefficient, and cost prohibitive in a pre-clinical trials and subsequent use in therapeutics. Chloroplast-derived ACE2 and Ang-(1-7) should reduce cost and facilitate oral delivery of these therapeutic proteins, thus making it attractive for clinical trials for above mentioned chronic diseases.

In Example 2, we described methods for enhancing the systemic and local activity of the protective axis of the RAS by oral delivery of ACE2 and Ang-(1-7) bioencapsulated in plant cells for conferring protection against endotoxin induced uveitis (EIU) and experimental autoimmune uveoretinitis (EAU). Both ACE2 and Ang-(1-7), fused with the non-toxic cholera B subunit B (CTB) were expressed in plant chloroplasts. The effects of orally delivered CTB-ACE2/Ang-(1-7) on EIU and EAU models in C57B6/J and B10.RIII mice respectively were examined. Increased levels of ACE2 and Ang-(1-7) were observed in circulation and retina after oral administration of CTB-ACE2/Ang-(1-7) leaf materials. Oral feeding of mice with bioencapsulated ACE2 or Ang-(1-7) significantly reduced LPS induced infiltration of inflammatory cells and expression of inflammatory cytokines in the eye; this treatment also dramatically decreased cellular infiltration, retinal vasculitis, damage and folding in EAU eyes. Thus, enhancing the protective axis of RAS by oral delivery of ACE2/Ang-(1-7) bioencapsulated in plant cells provide an innovative, more efficient and cost-effective therapeutic strategy for ocular inflammation such as uveitis and autoimmune uveoretinitis.

Definitions

As used herein, the terms “administering” or “administration” of an agent, drug, or peptide to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. The administering or administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, or topically. Administering or administration includes self-administration and the administration by another.

As used herein, the terms “disease,” “disorder,” or “complication” refers to any deviation from a normal state in a subject.

As used herein, by the term “effective amount” “amount effective,” or the like, it is meant an amount effective at dosages and for periods of time necessary to achieve the desired result.

As used herein, the term “inhibiting” or “preventing” means causing the clinical symptoms of the disease state not to worsen or develop, e.g., inhibiting the onset of disease, in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state.

As used herein, the term “expression” in the context of a gene or polynucleotide involves the transcription of the gene or polynucleotide into RNA. The term can also, but not necessarily, involves the subsequent translation of the RNA into polypeptide chains and their assembly into proteins.

A plant remnant may include one or more molecules (such as, but not limited to, proteins and fragments thereof, minerals, nucleotides and fragments thereof, plant structural components, etc.) derived from the plant in which the protein of interest was expressed. Accordingly, a composition pertaining to whole plant material (e.g., whole or portions of plant leafs, stems, fruit, etc.) or crude plant extract would certainly contain a high concentration of plant remnants, as well as a composition comprising purified protein of interest that has one or more detectable plant remnants. In a specific embodiment, the plant remnant is rubisco.

In another embodiment, the invention pertains to an administrable composition for treating or preventing pulmonary hypertension or pulmonary hypertension-induced lung pathophysiology. The composition comprises a therapeutically-effective amount of ACE2, Ang-(1-7), CTB-ACE2 or CTB-Ang-(1-7), or a combination thereof having been expressed by a plant and a plant remnant. The compositions of the invention may also be used to advantage to treat ocular inflammation.

Methods, vectors, and compositions for transforming plants and plant cells are taught for example in WO 01/72959; WO 03/057834; and WO 04/005467. WO 01/64023 discusses use of marker free gene constructs.

Proteins expressed in accord with certain embodiments taught herein may be used in vivo by administration to a subject, human or animal in a variety of ways. The pharmaceutical compositions may be administered orally or parenterally, i.e., subcutaneously, intramuscularly or intravenously, though oral administration is preferred.

Oral compositions of the present invention can be administered via the consumption of a foodstuff that has been manufactured with the transgenic plant producing the plastid derived therapeutic protein. The edible part of the plant, or portion thereof, is used as a dietary component. The therapeutic compositions can be formulated in a classical manner using solid or liquid vehicles, diluents and additives appropriate to the desired mode of administration. Orally, the composition can be administered in the form of tablets, capsules, granules, powders and the like with at least one vehicle, e.g., starch, calcium carbonate, sucrose, lactose, gelatin, etc. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, e.g., water, saline, dextrose, glycerol, ethanol or the like and combination thereof. In addition, if desired, the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants. In a preferred embodiment the edible plant, juice, grain, leaves, tubers, stems, seeds, roots or other plant parts of the pharmaceutical producing transgenic plant is ingested by a human or an animal thus providing a very inexpensive means of treatment of or immunization against disease.

In a specific embodiment, plant material (e.g. lettuce material) comprising chloroplasts capable of expressing ACE2, Ang-(1-7), CTB-ACE2 or CTB-Ang-(1-7), or a combination thereof, is homogenized, lyophilized and encapsulated. In one specific embodiment, an extract of the lettuce material is encapsulated. In an alternative embodiment, the lettuce material is powderized before encapsulation.

In alternative embodiments, the compositions may be provided with the juice of the transgenic plants for the convenience of administration. For said purpose, the plants to be transformed are preferably selected from the edible plants consisting of tomato, carrot and apple, among others, which are consumed usually in the form of juice.

According to another embodiment, the subject invention pertains to a transformed chloroplast genome that has been transformed with a vector comprising a heterologous gene that expresses a peptide as disclosed herein.

Of particular present interest is a chloroplast genome that has been transformed with a vector comprising a heterologous gene that expresses one or more polypeptides selected from ACE2, Ang-(1-7), CTB-ACE2 or CTB-Ang-(1-7), or a combination thereof. In a related embodiment, the subject invention pertains to a plant comprising at least one cell transformed to express a peptide as disclosed herein.

Reference to CTB and ACE2 or Ang-(1-7) sequences herein relate to the known full length amino acid sequences as well as at least 12, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250 or 265 contiguous amino acids selected from such amino acid sequences, or biologically active variants thereof. Typically, the polypeptide sequences relate to the known human versions of the sequences.

Variants which are biologically active, refer to those, in the case of oral tolerance, that activate T-cells and/or induce a Th2 cell response, characterized by the upregulation of immunosuppressive cytokines (such as IL10 and IL4) and serum antibodies (such as IgG1), or, in the case of desiring the native function of the protein, is a variant which maintains the native function of the protein. Preferably, naturally or non-naturally occurring polypeptide variants have amino acid sequences which are at least about 55, 60, 65, or 70, preferably about 75, 80, 85, 90, 96, 96, or 98% identical to the full-length amino acid sequence or a fragment thereof. Percent identity between a putative polypeptide variant and a full length amino acid sequence is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes).

Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active LecA polypeptide can readily be determined by assaying for native activity, as described for example, in the specific Examples, below.

Reference to genetic sequences herein refers to single- or double-stranded nucleic acid sequences and comprises a coding sequence or the complement of a coding sequence for polypeptide of interest. Degenerate nucleic acid sequences encoding polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 60, preferably about 75, 90, 96, or 98% identical to the cDNA may be used in accordance with the teachings herein polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of −12 and a gap extension penalty of −2. Complementary DNA (cDNA) molecules, species homologs, and variants of nucleic acid sequences which encode biologically active polypeptides also are useful polynucleotides.

Variants and homologs of the nucleic acid sequences described above also are useful nucleic acid sequences. Typically, homologous polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions: 2×SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2×SSC, 0.1% SDS, 50° C. once, 30 minutes; then 2×SSC, room temperature twice, 10 minutes each homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.

Species homologs of polynucleotides referred to herein also can be identified by making suitable probes or primers and screening cDNA expression libraries. It is well known that the Tm of a double-stranded DNA decreases by 1-1.5° C. with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973). Nucleotide sequences which hybridize to polynucleotides of interest, or their complements following stringent hybridization and/or wash conditions also are also useful polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2^(nd) ed., 1989, at pages 9.50-9.51.

Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20° C. below the calculated T_(m) of the hybrid under study. The T_(m) of a hybrid between a polynucleotide of interest or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962): T _(m)=81.5° C.−16.6(log₁₀[Na⁺])+0.41(% G+C)−0.63(% formamide)−600/l),

-   -   where l=the length of the hybrid in basepairs. Stringent wash         conditions include, for example, 4×SSC at 65° C., or 50%         formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C.         Highly stringent wash conditions include, for example, 0.2×SSC         at 65° C.

The Examples set forth below are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

Example 1 Creation of Transplastomic Plants Expressing CTB-ACE2 and CTB-Ang-(1-7)

In the present example, we describe a low cost oral delivery system for administering ACE2 or Ang-(1-7) and test its efficacy in an experimental model of PH. We took advantage of transplastomic technology which enables chloroplasts to generate high levels of therapeutic proteins within plant leaves.¹⁶⁻²¹ This technology presents minimal risk of human pathogen or endotoxin contamination, eliminates complex protein purification steps, and abolishes cold chain and sterile delivery requirements that are commonly associated with protein therapy.²²⁻²⁴ Efficacy of plant-based pharmaceuticals has been validated by the fact that FDA has recently approved the use of taliglucerase alfa (Trade name: Elelyso) in the treatment of Gaucher's disease.²⁵ This study provides evidence for the development of an oral delivery system to administer ACE2 and Ang-(1-7) using transplastomic technology, and demonstrates its efficacy in an established rat model of monocrotaline (MCT)-induced PH.

The following materials and methods are provided to facilitate the practice of Example 1.

Chloroplast Transformation Vector Construction and Regeneration of Transplastomic Lines

The cDNA for ACE2 (accession: NM_021804) was used as the template to clone CTB-ACE2 fusion gene into the intermediate vector. To clone CTB-Ang-(1-7) fusion gene, CTB-containing vector was used as template with a forward primer specific to CTB and a reverse primer specific to CTB but including nucleotide sequence corresponding to 7 amino acid of Ang-(1-7) peptide. The sequence-confirmed chimeric gene was cloned into chloroplast transformation vector, pLD.

Southern Blot Analysis

To investigate transgene integration and homoplasmy, Southern blot analysis was performed as previously described.¹ Total genomic DNA was digested with HindIIII, separated on a 0.8% agarose gel at 15 V overnight, transferred onto nylon membrane. The 0.8-kb flanking region probe was generated by digesting the pUC-CT vector DNA with BamHI and BglII. The probe was labeled with dCTP using Klenow fragment (Promega M220A) and random primers (Promega C1181). After labeling the probe, the blotted membrane was hybridized with hybridization solution [0.5 M phosphate buffer pH 7.2, 1 mM EDTA pH 8.0, 7% (w/v) SDS, 1% (w/v) bovine serum albumin] at overnight at 65° C. then washed with 2×SSC, 0.2% SDS for 30 min once and 1×SSC, 0.1% SDS for 15 min twice each. Radioisotope-labelled blots were exposed to X-ray film at −80° C. for 8 h.

Quantification of CTB-ACE2 and CTB-Ang-(1-7) Fusion Protein

Leaves ground in liquid nitrogen was resuspened in extraction buffer [100 mM NaCl, 10 mM EDTA, 200 mM Tris-Cl pH 8.0, 0.1% (v/v) Triton X-100, 400 mM sucrose, 2 mM PMSF, and proteinase inhibitor cocktail] in a ratio of 100 mg to 300 μL, then vigorously mixed using vortex (˜30 s) and sonicated twice (pulse on for 5 s and pulse off for 10 s). Homogenate protein was quantified using Bradford assay. For the quantification of CTB-ACE2 protein, ELISA was performed. Ninety-six-well plates were coated with serially diluted CTB standard (25-12.5-6.25-3.13-1.56-0.781-0.391-0.195 pg/μL; Sigma C9903) and CTB-ACE2 proteins (4,000-8,000-16,000) in bicarbonate buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, 3.08 mM NaN₃, pH 9.6), then incubated overnight at 4° C. After washing the plates with 1× phosphate buffered saline (Fisher IC-N2810307) containing 0.05% (v/v) Tween 20 (1×PBST), the coated plates were blocked with 1×PBST containing 3% skim milk (PTM) for one and half hr at 37° C. and incubated with rabbit anti-CTB polyclonal antibody (1:10,000 in PTM; GenWay 18-511-245283) overnight at 4° C. The plates were followed by incubating with goat anti-rabbit IgG-HRP secondary antibody (1:4,000 in PTM; Southern Biotechnology 4030-05) for one and half hr at 37° C. after washing with 1×PBST thrice. The antibody-bound plates were washed with 1×PBST thrice and 1×PBS once before adding the 100 μL of tetramethyl benzidine (TMB) solution substrate (American Qualex Antibodies UCFL-C5801). The reaction was stopped by adding the 50 μL of 2N H₂SO₄ and read on a plate reader at 450 nm. CTB-Ang-(1-7) was quantified using western blot and densitometric analysis. Total homogenate protein (0.5 μg) and CTB standards (3, 6, and 9 ng) was separated on SDS-PAGE and transferred to nitrocellulose membranes. Rabbit anti-CTB polyclonal antibody (1:10,000 in PTM; GenWay) and goat anti-rabbit IgG-HRP secondary antibody (1:4,000 in PTM; Southern Biotechnology) was used to detect the fusion proteins. SuperSignal West Pico Chemiluminescent Substrate (Pierce 34080) was used for autoradiographic detection. Then the developed films were analyzed by densitometry using Image J (IJ 1.46r; NIH). The known amounts of CTB standard were plotted and then the protein samples were interpolated on the graph. For the separation of proteins under non-reducing conditions, proteins were extracted as described above. The extracted proteins were combined with tricine sample buffer (Bio-Rad 161-0730), in the absence of reducing agents and without boiling prior to running on SDS-PAGE gels.

GM1 Binding Assay

To evaluate pentameric structure, GM1 binding assay was performed. Ninety-six-well plates were coated with monosialoganglioside-GM1 (Sigma G-7641) (3.0 μg/mL in bicarbonate buffer: 15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) overnight at 4° C. The plates were washed with PBST thrice and blocked with PTM. After washing the plates with 1×PBST, homogenate plant protein was diluted to concentration of 0.1 μg/μL with the same plant extraction buffer and incubated in the GM1 coated plates overnight at 4° C., along with CTB (1 ng/μL, Sigma), bovine serum albumin (1% w/v BSA) and untransformed wild type plant protein (0.1 μg/μL) as controls. The plates were blocked with PTM for one and half hr at 37° C. After discarding the PTM, rabbit anti-CTB polyclonal antibody (1:10,000 in PTM; GenWay) was incubated overnight at 4° C. Following washing three times with 1×PBST, goat anti-rabbit IgG-HRP secondary antibody (1:4,000 in PTM; Southern Biotechnology) was incubated for one and half hr at 37° C. The plates were washed with PBST thrice and with PBS once and 100 μL of tetramethyl benzidine (TMB) solution substrate (American Qualex Antibodies UCFL-C5801) was added to the wells and incubated under dark for 5 min. The reaction was stopped by adding 50 μL of 2N H₂SO₄, and read the absorbance at 450 nm using plate reader (Bio-rad Model 680).

Lyophilization

Frozen leaf tissues stored at −80° C. were crumbled into small pieces and transferred to 200 ml containers and sealed with porous 3M Millipore Medical Tape. The plant samples were freeze-dried in vacuum at −52° C. at 0.036 mBar for three days, with the aid of VirTis BenchTop 6K freeze dryer system. Lyophilized leaf material was stored in sealed container at room temperature with silica gel.

PH Study Design

We used the monocrotaline (MCT) animal model of PH to evaluate the therapeutic efficacy of oral feeding of ACE2, Ang-(1-7) or their combination against disease pathogenesis. Animals were randomly assigned to respective experimental groups based on their body weights at the time of MCI administration. The study design consisted of prevention and reversal protocols. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida and complied with National Institutes of Health guidelines.

Gavage Feeding of MCT Rats with Bioencapsulated ACE2 or Ang-(1-7)

8-week-old male Sprague Dawley rats (Charles River Laboratories) were injected with a single subcutaneous dose of MCT (50 mg/kg, Sigma Aldrich, USA). Control animals received an equivalent amount of sterile saline (˜500 μL). For the prevention protocol, a subset of MCT animals was simultaneously orally gavaged with wild type leafy material, bioencapsulated ACE2 or Ang-(1-7) [500 mg, twice daily in sterile phosphate-buffered saline (PBS)] for a period of 28 days. For the reversal protocol, ACE2, Ang-(1-7) or their combination [500 mg or 250 mg each of ACE2 and Ang-(1-7)] was gavage-fed after 2 weeks of MCT administration and continued for additional 15 days.

Echocardiography Measurement

Four weeks after MCT injection, transthoracic echocardiography was performed using GE vivid7 ultrasound machine with a 12-MHz transducer (GE Healthcare, NJ, USA). Rats were anesthetized with the 2% isoflurane-oxygen mixture. M-mode echocardiography was measured at the parasternal short-axis view at the level of papillary muscles. Left ventricular ejection fraction (LVEF) was calculated from the M-mode. Further, at this view, right and left ventricular end diastolic area (RVEDA and LVEDA) and right ventricular ejection fraction (RVEF) were also measured. Pulsed Doppler recordings performed at the parasternal short-axis view at the base of the heart to measure the right ventricle outflow tract (RVOT V_(max)). ECG was recorded simultaneously for all the assessments. All the recordings were performed in triplicates. Ejection fraction was obtained from both right and left ventricles and was represented as the ratio between right and left ventricle. Similarly, end diastolic area was also represented as the ratio between right and left ventricle. Blood flow at the right ventricular outflow tract was represented as RVOT Vmax (m/s). Three consecutive cycles from each recording (totally 9 cycles) were averaged to assess each parameter. Following the echocardiographic measurements, animals were subjected to hemodynamic measurements.

Right Ventricular Systolic Pressure (RVSP) Measurements

The RVSP was measured in anesthetized animals [subcutaneous injection of a mixture of ketamine (30 mg/Kg) and Xylazine (6 mg/Kg)] using a fluid-filled silastic catheter, which was inserted inside the right descending jugular vein and advanced to the right ventricle. The catheter was connected to a pressure transducer that was interfaced to a PowerLab (AD Instruments, USA) signal transduction unit. The waveform was used to confirm the positioning of the catheter in the right ventricle. RVSP, +dP/dt, −dP/dt and right ventricular end diastolic pressure (RVEDP) were obtained using the Chart program supplied along with the PowerLab system. For both prevention and reversal protocols RVSP was measured after 4 weeks of MCT-challenge.

Hypertrophy and Histological Analysis

Following RVSP measurements, a thoracotomy was performed, and after exsanguination, the heart and lungs were removed en bloc. To calculate right ventricular hypertrophy (RVH), the wet weight of RV and left ventricle plus ventricular septum (LV+S) was determined. RVH was expressed as the ratio of RV/[LV+S] weights. The RV was further processed for histological analysis of collagen content. Briefly, RV was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 μm and stained with picro-sirius red. Interstitial fibrosis was determined at 100× magnification using the ImageJ program from National Institutes of Health, as previously described.² A minimum of 5-8 separate images from different (non-overlapping) regions of the right ventricle were obtained. The results for each animal were then averaged for subsequent statistical analysis. To carry out histological examination of the lung, the left lung alone was perfused with PBS followed by 10% neutral buffered formalin. For measuring pulmonary medial wall thickness, 5 μm thick lung sections were cut paraffin embedded and stained for a-smooth muscle actin (1:600, clone 1A4, Sigma Aldrich, USA). Vessels with an external diameter of <50 μm were considered to measure the medial wall thickness. For each rat, around 10 vessels were counted and the average was calculated. The percent medial wall thickness was calculated using the formula: % Medial wall thickness=[(medial thickness×2)/external diameter]×100 (n=5 rats per group) Media thickness was defined as the distance between the lamina elastica interna and lamina elastica externa.

Real-Time RT-PCR Analysis

Semi-quantitative real time RT-PCR was used to determine mRNA levels of the renin-angiotensin system components viz. ACE, ACE2, AT1R, and AT2R, and pro-inflammatory cytokines (PICs) viz. Tumor Necrosis Factor-alpha (TNF-α), Transforming Growth Factor-beta (TGF-β) and toll-like receptor-4 (TLR-4) as described previously.² Total RNA isolation, cDNA synthesis and RT-PCR were performed as previously described. In brief, total RNA was isolated from punched tissues using TRIzol reagent (Invitrogen, USA) according to the manufacturer's specifications. The RNA concentration was calculated from the absorbance at 260 nm and RNA quality was assured by 260/280 ratio. Only RNA samples exhibiting an absorbance ratio (260/280) of >1.6 were used for further experiments. The RNA samples were treated with DNase I (Ambion, USA) to remove any genomic DNA. First strand cDNA was synthesized from 2 μg RNA with iScript cDNA synthesis kit (Bio-Rad, USA). Real-time RT-PCR was performed in 384-well PCR plates using iTaq SYBR Green Super mix with ROX (Bio-Rad) in triplicate using the ABI Prism 7900 sequence detection system (Applied Biosystems, USA). The PCR cycling conditions were as follows: 50° C. for 2 min, 95° C. for 3 min, followed for 45 cycles (15 s at 95° C., and 1 min at 60° C.). To confirm the specific PCR product, a dissociation step (15 s at 95° C., 15 s at 60° C., and 15 s at 95° C.) was added to check the melting temperature. Gene expression was measured by the ΔΔCT method and was normalized to 18 S mRNA levels. The data are presented as the fold change of the gene of interest relative to that of control animals.

Measurement of Ang-(1-7):

Circulating levels of Ang-(1-7) were measured using a commercially available EIA kit from Bachem Laboratories as per manufacturer's instructions.

Statistics

Prism 5 (GraphPad) was used for all analyses. Values are presented as means±SEM. Data were analyzed using one-way ANOVA followed by the Newman-Keuls test for multiple comparisons. P values less than 0.05 were considered statistically significant.

Results

Creation and Characterization of CTB-ACE2 and CTB-Ang-(1-7) Expressed in Plant Chloroplasts:

The native human ACE2 cDNA and synthetic Ang-(1-7) DNA sequences were cloned into the chloroplast transformation vector (pLDutr) (FIG. 1A). For efficient delivery of the proteins into circulation, a carrier protein, cholera non-toxic B subunit (CTB), was fused to the N-terminal of both therapeutic proteins (FIG. 1A), which facilitates their transmucosal delivery by binding to monosialotetrahexosylganglioside receptors (GM1) present on the intestinal epithelial cells. Hinge (Gly-Pro-Gly-Pro; SEQ ID NO: 1) and furin cleavage site (Arg-Arg-Lys-Arg; SEQ ID NO: 2) were placed between CTB and therapeutic proteins (FIG. 1A) to eliminate steric hindrance and aid systemic release of these therapeutic proteins after they are internalized via ligand-receptor complex formation on the surface of epithelial cells. The expression of the fusion genes was driven by light regulated strong chloroplast psbA promoter and the transcripts were stabilized by placing the psbA UTR at the 3′ end of the fusion genes (FIG. 1A). To select the chloroplast transformed with the fusion genes, aminoglycoside-3″-adenylyl-transferase gene (aadA), driven by the chloroplast ribosomal RNA promoter (Prrn), was incorporated into the expression cassette to confer the transformants resistance to spectinomycin (FIG. 1A). This expression cassette was flanked by DNA sequences of isoleucyl-tRNA synthetase (trnI) and alanyl-tRNA synthetase (trnA) genes, identical to the native chloroplast genome at both flanks (FIG. 1A). The flanking sequences serve to facilitate transgene integration into the chloroplast genome (FIG. 1A) via double homologous recombination. Chloroplast transformation vectors expressing the ACE2 and Ang-(1-7) genes were coated onto gold particles and delivered into chloroplasts using the biolistic particle delivery system.²⁶ The bombarded plant leaves were then grown on spectinomycin containing plant regeneration media. The shoots regenerated from the media were investigated for the site specific integration of the transgenes into chloroplast genome and homoplasmy of the transgenes (absence of untransformed genomes) using Southern blot analysis with the radioisotope-labelled probe spanning trnI and trnA flanking sequences.²⁶ HindIII-digested chloroplast genomic DNA from three independent transplastomic lines for each transplastomic line showed two hybridizing fragments at 8.59 and 3.44 kb for CTB-ACE2 due to an internal Hind III site of ACE2 (FIG. 1A) and a fragment at 9.71 kb for CTB-Ang-(1-7), which confirm the absence of untransformed chloroplast genomes (FIGS. 1B and 1C). Thus, stable integration of the transgenes was confirmed and the homoplasmic lines were used for further studies. The confirmed homoplasmic lines were multiplied using another round of antibiotic selection under aseptic conditions. Then they were cultivated in a controlled greenhouse for increasing biomass. CTB-ACE2 expression varied between 1.69% and 2.14% of the total leaf proteins (TLP) (FIG. 1D), depending upon the harvest time because this transgene is regulated by light via the chloroplast psbA promoter. Similarly, the expression level of CTB-Ang-(1-7) varied between 6.0% and 8.7% of TLP (FIG. 1E), at different durations of illumination, reaching maximum expression at the end of the day. Hence, for performing in vivo experimental studies, the therapeutic leaf materials were harvested at 6 pm and powdered in liquid nitrogen.

Both the therapeutic proteins were fused to the transmucosal carrier, CTB. The B subunit has a single intra-subunit disulfide bond which stabilizes the CTB monomer.²⁵ The monomers then assemble to form ring-shaped pentameric structure via inter-subunit interactions including hydrogen bonds, salt bridges, and hydrophobic interactions. Upon oral administration, only the pentameric form of CTB binds to the gut epithelial GM1 receptor for internalization.²⁷ Hence, we investigated the proper formation of pentameric structure of the CTB fused proteins and their binding affinity to GM1 receptor using GM1-ELISA. The binding affinity between CTB pentamers and the receptor was measured spectrophotometrically as a function of absorbance at 450 nm. The therapeutic proteins from the fresh leaf materials showed comparable absorbance to CTB (FIG. 1F), confirming that chloroplasts form disulfide-bridges, fold, and assemble these fusion proteins. We also lyophilized the leaves expressing ACE2 and Ang-(1-7), and evaluated their affinity to the GM1 receptor (FIG. 1F). Lyophilization not only maintained proper folding, disulfide bond and pentamer assembly but also facilitated long-term storage at room temperature (FIG. 1F). Furthermore, the Western blot assay performed under non-reducing conditions without DTT and boiling showed that there was no monomeric form or cleaved fragments of CTB-Ang-(1-7) (FIG. 1G). In the Western blot image, the major bands for pentameric assembly of CTB were detected around ˜50 kDa (FIG. 1G, arrow head) and the expected bands for pentameric assembly of CTB-Ang-(1-7) were detected (FIG. 1G, arrow). Therefore, these results confirm that the therapeutic proteins expressed in chloroplasts exist in an intact and pentameric form.

Oral Feeding of Bioencapsulated ACE2 or Ang-(1-7) Prevents MCT-Induced PH

Oral gavage of the frozen powdered leaves (500 mg in sterile phosphate buffered saline) from untransformed wild type, CTB-ACE2 or CTB-Ang-(1-7) transplastomic plants was performed twice daily for 4-weeks in MCT-challenged rats. MCT injection caused robust elevation in right ventricular systolic pressure (RVSP; FIG. 2A) that was associated with the development of right ventricular hypertrophy (RVH) (FIG. 2B). In contrast, MCT animals gavaged with either ACE2 or Ang-(1-7) showed considerable reduction in RVSP and RVH (FIGS. 2A and 2B). Furthermore, measurement of hemodynamic parameters in MCT animals revealed increases in right ventricular end diastolic pressure (RVEDP; 153%), +dP/dt (88%) and −dP/dt (107%). Conversely, treatment with ACE2 or Ang-(1-7) restored all these parameters to near-control levels (FIGS. 2C, 2D, and 2E). Echocardiography of MCT rats revealed an increase in the ratio of right ventricle to left ventricle end diastolic area (RV/LV EDA), implying dilation of the right heart (FIG. 2F, and FIGS. 3A and 3B), which was accompanied with a decrease in ejection fraction (EF), measured as a ratio of RV/LV EF (FIG. 2G and FIGS. 3C and 3D). In addition, the pulsed Doppler blood flow measurement revealed decreased flow rate in the right ventricle outflow tract (RVOT) (FIG. 2H). Furthermore, video of the echocardiography revealed maladaptive structural remodeling in MCT rat hearts as compared with controls. However, oral delivery of ACE2 or Ang-(1-7) exhibited improved cardioprotective effects. Both ACE2 and Ang-(1-7) were effective in decreasing RV dilation (FIG. 2F), increasing EF (FIG. 2G) and preventing MCT-induced decrease in RVOT blood flow (FIG. 2H). These beneficial effects were associated with reduced cardiac remodeling as evidenced by echocardiography videos. Concurrently, RV fibrosis and pulmonary vessel wall thickness were also decreased (FIGS. 6A and 6B). Oral ACE2 feeding was associated with ˜37% increase in circulating ACE2 activity as compared with MCT alone rats (FIG. 6C) and a two-fold increase in circulating levels of Ang-(1-7; FIG. 4). Interestingly, ACE2 or Ang-(1-7) did not alter the basal systemic blood pressure (SBP: Control, 120±5; MCT, 123±7; MCT+ACE2, 118±2; MCT+Ang-(1-7), 116±4; n=5/experimental group).

Oral ACE2/Ang-(1-7) Treatment Arrests the Progression of Established PH

We next tested whether oral feeding of ACE2 or Ang-(1-7) after the initiation of PH could arrest the disease-progression. We observed that two-weeks of MCT challenge induces significant elevation in RVSP (>45 mmHg) as compared with controls (FIG. 5A) Hence, for this study, oral therapy was initiated after two-weeks of MCT challenge, and the treatment continued for additional 15-days. This regime of treatment with ACE2 or Ang-(1-7) inhibited further elevation in MCT-induced RVSP and RVH (FIGS. 7A and 7B), and was associated with increased circulating levels of Ang-(1-7; FIG. 4). Improvements in hemodynamic parameters with regard to lowering RVEDP, decreasing +dP/dt, and reducing −dP/dt were also observed (FIGS. 5B and 5D). In addition, ACE2/Ang-(1-7) therapy decreased RV dilation (FIG. 7C, and FIGS. 3A and 3B) and increased RVEF (FIG. 7D and FIGS. 3C and 3D), which was supported by echocardiography video. Subsequently, blood flow in the RVOT was also improved (FIG. 7E). Finally, RV fibrosis and pulmonary vessel wall thickening were significantly attenuated in ACE2/Ang-(1-7) treated animals (FIGS. 7F and 7G).

Combination Therapy with Oral ACE2 and Ang-(1-7) Feeding Rescues Established PH

Next, we evaluated the effects of a combination therapy with ACE2 and Ang-(1-7), wherein 500 mg or 250 mg each of ACE2 and Ang-(1-7) plant material was combined. Reversal protocol was followed for this study, wherein the combination therapy was initiated after two-weeks of MCT challenge, and the treatment continued for the next 15 days. As expected, we observed better protective effects with the 500 mg combination. This combination showed 18% more reduction in RVSP and 25% additional decrease in RV/(LV+S) ratio when compared with the 250 mg combination (FIGS. 8A and 8B). Similarly, enhanced beneficial effects of the 500 mg combination were observed for other hemodynamic parameters such as RVEDP, +dP/dt, and −dP/dt (FIGS. 8C to 8E). Both doses of the combination therapy were effective in decreasing RV dilatation (FIG. 8F, and FIGS. 3A and 3B), and increasing EF (FIG. 5G, and FIGS. 3C and 3D), which was accompanied by greater RVOT blood flow (FIG. 8H). All these observations were supported by echocardiography video. Consistent with this were the improvement in RV fibrosis and pulmonary vessel wall thickness following combination therapy (FIGS. 9A and 9B).

Beneficial Effects of ACE2/Ang-(1-7) Oral Therapy Involve Inhibition of Pro-Inflammatory Cytokines and Autophagy

We demonstrate herein that oral delivery of ACE2 or Ang-(1-7) corrects RAS imbalance and inhibit pro-inflammatory cytokines. Data in FIG. 10 support this hypothesis. MCT rats revealed increased pulmonary mRNA levels of ACE and AT1R (FIGS. 10A and 10D), which resulted in 8-fold and 4-fold increases in the ACE/ACE2 and AT1R/AT2R ratios respectively (FIGS. 10C and 10F). Conversely, mRNA levels of ACE2 and AT2R were increased, while that of AT1R was decreased in the ACE2 or Ang-(1-7) fed MCT rats, resulting in decreased ACE/ACE2 and AT1R/AT2R ratios. Furthermore, MCT-challenged animals showed increased mRNA levels of TNF-α (4-fold), TGF-β (4-fold) and TLR-4 (5-fold), all of which were markedly reduced by ACE2 or Ang-(1-7) treatment (FIGS. 10G, 10H and 10H). Recent reports indicate that the autophagic protein degradation pathway is activated in MCT-challenged animals.²⁸ Accordingly, we observed that the lung LC3B-II protein, an autophagy marker, was significantly increased in MCT-challenged rats (FIG. 10J). However, ACE2 or Ang-(1-7) decreased LC3B-II levels, implying inhibition of autophagy. Similar results with respect to RAS modulation, anti-inflammatory properties and inhibition of autophagy were observed in the reversal protocol with monotherapy [either ACE2 or Ang-(1-7)] or combination therapy (FIGS. 11A to 11J).

Discussion

Human ACE2 and Ang-(1-7) has been expressed within plant chloroplasts using transplastomic technology. Oral administration of transplastomic plant material to rats attenuates PH. While previous genetic interventions with ACE2/Ang-(1-7) have demonstrated beneficial effects in animals,^(11,12) there are several challenges that limit the clinical development of such approaches. The incidence of PH is increasing among the elderly global population, necessitating affordable medication for the masses. While drugs made in plant cells have been approved by the FDA and are currently marketed,²⁵ targeted gene therapy is still in the experimental stage and far away from clinical applications. Even if gene therapy is approved as a valid approach, it would still be available to <1% of the global population due to the limited expertise available in hospitals for gene therapy. In contrast, oral delivery of capsules containing therapeutic proteins produced in plant chloroplasts is feasible and very much affordable. Accordingly, drug delivery is as important as drug discovery and this study focuses on the development of a novel low cost delivery system for administering therapeutic proteins like ACE2/Ang-(1-7), which have been found to be effective against experimental models of lung diseases, but not yet clinically approved. Injectable delivery of ACE2/Ang-(1-7) poses some unique challenges with respect to cost of manufacturing, protein stability, cold storage, shelf life, sterile delivery and requirement of health professionals/hospitals for their administration. Most of these concerns are easily eliminated by orally delivering therapeutic proteins bioencapsulated in plant cells. Currently produced injectable protein drugs are not affordable to more than half of the global population, despite decades of optimization of their process development. By developing an oral delivery system for administering ACE2 and Ang-(1-7), as reported here, we have made a tremendous advancement to move the field for the treatment of pulmonary diseases forward.

ACE2 and Ang-(1-7) were expressed in plant chloroplasts as fusion proteins with CTB. Though Ang-(1-7) is not a gene product, a synthetic gene encoding for Ang-(1-7), was used in this study.²⁹ We have used CTB as the transmucosal carrier to facilitate the uptake of ACE2 and Ang-(1-7) into circulation. Both CTB fusion proteins are disulfide bonded, form pentamers and properly folded, as observed for other CTB fusion proteins.^(17,18) CTB is an approved adjuvant³⁹ that has been used in several clinical settings. Administration of CTB fused antigen (BD peptide) in humans with autoimmune eye disorders induced immunological tolerance by suppressing abnormal T cell reactivity against the peptide.⁴⁰ Also, immune suppression to autoantigens (pro-insulin and factor IX) linked to CTB have been observed in animal studies following oral administration.^(19,20) Likewise, other studies have shown immune-suppressive effects when CTB was fused to autoimmune or allergic causative agents.⁴¹ The GM1 receptors present on intestinal epithelial cells make CTB the most appropriate carrier for transporting therapeutic proteins into systemic circulation as this receptor is widely distributed over the intestinal mucosa^(42, 43) with a rapid turnover rate.⁴⁴

The half-life of native Ang-(1-7) is very short.^(45,46) However, in this study, the stability of Ang-(1-7) was found to increase in sera. In plant cells, CTB stabilizes Ang-(1-7) by formation of pentamers (FIG. 1G), and thus confers protection from plant proteases. However, only monomers are observed in sera after delivery into the sera. While furin cleavage site (NH2-R—R—K—R—COOH) should facilitate removal of CTB, efficiency of cleavage depends upon the flanking amino acid sequence of the fused protein.⁴⁷ Ang-(1-7) fused to CTB didn't provide optimal furin cleavage site because it is not flanked by furin preferred basic amino acids at N-terminal side and serine-valine at C-terminal side. Therefore, it is anticipated that furin cleavage will not be rapid or efficient. This offers greater N-terminal protection to Ang-(1-7) and extends its stability for several hours in the sera as compared with injectable Ang-(1-7). Actually, chronic treatment with bioencapsulated Ang-(1-7) showed significant increases of the circulating levels of the peptide (FIG. 4), which suggests that an oral gavage twice daily of bioencapsulated Ang-(1-7) results in sustained elevated plasma levels of Ang-(1-7) in the treated animals. Ang-(1-7) concentration in frozen leaf materials was found to be 584 μg/g, which translates to 292 μg in 500 mg. This dose compares well with previous studies, wherein, 750-1000 μg/Kg of Ang-(1-7) peptide was administered to rats (˜300 ug per 300 gms rat).

Oral feeding of bioencapsulated ACE2 or Ang-(1-7) prevents the development, and most importantly, retards the progression of MCT-induced PH. An upregulation of the deleterious ACE-AngII-AT1R axis and downregulation of the protective ACE2-Ang-(1-7)-Mas axis contributes to PH pathogenesis.⁴⁸ Thus, maintaining equilibrium between these two axes is crucial for preserving pulmonary vascular homeostasis. Oral delivery of ACE2 or Ang-(1-7) increased ACE2/ACE and decreased AT1R/AT2R ratios, signifying improvement in pulmonary RAS balance. In addition, the ACE2-fed MCT rats prevented the decrease in serum ACE2 activity observed in PH animals. Also, about 2-3 fold increase in the circulating levels of Ang-(1-7) in ACE2 treated animals was observed, which could possibly contribute to the protective effects (FIG. 4). ACE2 fed rats exhibited 37% increase in the enzymatic activity as compared with MCT alone animals. This increase in disease treated animals restored the enzymatic activity of circulating ACE2 to that of normal healthy rats. Most importantly, this increase was sufficient to exert beneficial effects against PH pathophysiology. Previous experimental studies have shown that exogenous administration of recombinant ACE2 increases serum ACE2 activity to exert therapeutic efficacy in several disease models.⁴⁹⁻⁵¹ Increasing serum ACE2 levels is also clinically significant since abnormally low levels of serum ACE2 have been associated with PH.¹⁰ Surprisingly, pulmonary ACE2 mRNA levels were increased with oral ACE2 feeding. We speculate this increase to be a positive feed forward mechanism, since similar increases in ACE2 have been reported previously.^(14,15) Also, AT2R levels were increased with ACE2 treatment, which is consistent with previous studies showing a protective role of this receptor in cardiopulmonary disease.⁵² Furthermore, the favorable RAS modulation by ACE2 or Ang-(1-7) was associated with reduced lung inflammatory cytokines. Pro-inflammatory cytokines contribute to thickening of the pulmonary arterioles leading to heightened pulmonary pressure.⁵³ In line with these findings, we observed marked increases in vessel wall thickness in MCT-challenged animals. However, ACE2 or Ang-(1-7) treatment significantly inhibited medial wall thickness. The observed effects of ACE2/Ang-(1-7) could be attributed to reduction in pro-inflammatory cytokines as well as direct anti-proliferative actions on the vascular smooth muscle cells, a contention supported by earlier studies.⁵⁴ Recent studies have implicated autophagy in PH.²⁶ We observed an increase in LC3B-II, an autophagy marker, in MCT rats, which was significantly decreased with ACE2 or Ang-(1-7) treatment.

Collectively, the aforementioned findings indicate that oral delivery of ACE2 or Ang-(1-7) corrects a dysregulated pulmonary RAS, reduces inflammation, decreases vascular remodeling and inhibits autophagy to exert lung-protective effects. Importantly, ACE2/Ang-(1-7) treatment did not lower basal systemic blood pressure, which is important since induction of systemic hypotension can be detrimental in patients with PH. Similar phenomenon has also been observed in other studies, wherein, chronic administration of Ang-(1-7) fails to decrease systemic BP in a variety of models of hypertension⁵⁵⁻⁵⁷. One possibility may be related to pulmonary vasculature being more sensitive to Ang-(1-7) or that abundant receptors for Ang-(1-7) are present on the pulmonary vessels. Furthermore, a combination of ACE2 and Ang-(1-7) treatment produced beneficial effects on the cardiopulmonary system. We observed that the higher dose combination yielded better effects than the lower dose.

Of particular interest are the cardioprotective effects of oral ACE2/Ang-(1-7) therapy. Sustained pressure overload on the right heart induces ventricular remodeling and dysfunction.⁵⁸ Echocardiography of MCT rats revealed prominent structural changes in the heart. The RV assumed a round shape, with a shift in the intraventricular septum causing RV dilation with reduced EF. In addition, the pulmonary artery flow was significantly lowered in the MCT group. All these changes were associated with development of RVH, increased interstitial fibrosis and cardiac dysfunction. However, ACE2 or Ang-(1-7) treatment restored normal heart structure, inhibited RV dilatation and improved EF. Also, RVH and interstitial fibrosis were significantly reduced, along with preserved cardiac function. Moreover, the combination therapy with ACE2 and Ang-(1-7) was found to exert superior cardioprotective effects.

REFERENCES FOR EXAMPLE 1

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Example 2 Oral Delivery of ACE2/Ang-(1-7) Bioencapsulated in Plant Cells Protects Against Experimental Uveitis and Autoimmune Uveoretinitis

The following materials and methods are provided to facilitate the practice of Example II.

Chloroplast Transformation Vector Construction and Regeneration of Transplastomic Lines

Performed as described above in Example I.

Animals and Experimental Procedures

Wild-type C57B1/6J mice (6-8 weeks old)) and B10.RIII mice (8-10 weeks old) were purchased from Jackson Laboratories (Bar Harbor, Me.) and maintained at the Animal Care Service at the University of Florida. All procedures adhered to the ARVO statement for the use of Animals in Ophthalmic and Vision Research, and the protocol was approved by the Animal Care and Use Committee of the University of Florida. The animals were fed standard laboratory chow and allowed free access to water in an air-conditioned room with a 12-12-hr light dark cycle.

The mice were divided in three groups for EIU model and orally gavaged with control (untransformed wild-type, WT) tobacco leaves, CTB-ACE2, and CTB-Ang-(1-7) expressing transplastomic tobacco leaves. The mice were given ˜500 mg of the specified tobacco leaf material suspended in sterile PBS, by careful gavage into the hypopharynx twice in a day for 5 days. For preparation of the gavage material, leaves were frozen and ground in liquid nitrogen. EIU was induced by a single intravitreal injection of Escherichia coli LPS (25 ng/eye) (Sigma-Aldrich, Inc., St. Louis, Mo.) dissolved in sterile pyrogen-free saline, on the fifth day of feeding. All animals were anesthetized and pupils were dilated before intraocular injections. Each experimental group included at least 4-6 animals and each experiment was performed at least twice.

For the EAU model, the mice were divided in three groups and orally gavaged with ˜500 mg of the control wild-type tobacco leaves, CTB-ACE2, CTB-Ang-(1-7) expressing transplastomic tobacco leaves once daily for 15 days. EAU was induced by active immunization with ˜50 μg of IRBP (161-180) (SGIPYIISYLHPGNTILHVD) (Genscript, Piscataway, N.J.) with CFA (Sigma-Aldrich, Inc., St. Louis, Mo.) (1:1 vol/vol) subcutaneously, on the second day of feeding. Each experimental group included at least 4-6 animals and each experiment was performed at least twice to ensure reproducibility.

Different Doses of Lyophilized CTB-ACE2 Plant Cell Oral Gavage and Induction of EIU

In another approach, the mice were divided in three groups and orally gavaged with varying dosage of lyophilized CTB-ACE2 expressing transplastomic tobacco leaves. The mice were given 12.5 mg, 25 mg and 50 mg of the specified lyophilized plant cells suspended in sterile PBS, by careful gavage into the hypopharynx once in a day for 4 days EIU was induced by a single intravitreal injection of Escherichia coli LPS (25 ng/eye) (Sigma-Aldrich, Inc., St. Louis, Mo.) dissolved in sterile pyrogen-free saline, on the fourth day of feeding. All animals were anesthetized and pupils were dilated before intraocular injections. Each experimental group included at least 4-6 animals and each experiment was performed at least twice.

Delaying of Oral Gavage and Induction of EAU

The mice were divided in three groups and orally gavaged with ˜50 mg of the lyophilized plant cells expressing CTB-Ang-(1-7) once daily. The treatment with CTB-Ang-(1-7) started at day 5 and day 10 after IRBP injection to induce EAU, and continued daily till day 14. EAU was induced by active immunization with ˜50 μg of IRBP (161-180) (SGIPYIISYLHPGNTILHVD; SEQ ID NO: 22) (Genscript, Piscataway, N.J.) with CFA (Sigma-Aldrich, Inc., St. Louis, Mo.) (1:1 vol/vol) subcutaneously. Each experimental group included at least 4-6 animals and each experiment was performed at least twice to ensure reproducibility.

Histopathological Evaluation

The EIU mice were euthanized 24 hr after LPS injection and the eyes were enucleated immediately and fixed in 4% paraformaldehyde freshly made in PBS overnight at 4° C. and processed for paraffin embedding and sections. Sagittal sections (4 μm) from every 50 μm were cut and stained with hematoxylin and eosin (H&E). The anterior and posterior chambers were examined under light microscope and the infiltrating inflammatory cells were counted in a masked fashion. The number of infiltrating inflammatory cells in five sections per eye was averaged and recorded. EIU clinical data shown were representatives of three sets of experiments.

The EAU mice were euthanized and the eyes were harvested on 14th day after immunization, followed by fixation, paraffin embedment and stained with H&E. The severity of EAU was evaluated in a masked fashion on a scale of 0-4 using previously published criteria based on the number, type and size of lesions [32] and the inflammatory cells were counted as described above. EAU clinical data shown were representatives of two sets of experiments, 5 animals each experimental group.

ACE2 Activity Assay

Representative retinas from each group of mice were dissected and homogenized by sonication in ACE2 assay buffer. The ACE2 activity assay was performed using 100 μg of retinal protein in black 96-well opaque plates with 50 μM ACE2-specific fluorogenic peptide substrate VI (R&D Systems, Inc., Minneapolis, Minn.) in a final volume of 100 μl per well reaction mixture. The enzymatic activity was recorded in a SpectraMax M3 fluorescence microplate reader (Molecular Devices, LLC, Sunnyvale, Calif.) for 2 hr with excitation at 340 nm and emission at 400 nm as described previously [10]. For the sera samples, 10 μl sera were used in a 100 μl reaction. All measurements were performed in duplicate and the data represent the mean of three assay results.

Ang-(1-7) Estimation by Enzyme Immunoassay (EIA)

The level of Ang-(1-7) in plasma and retina were measured using a commercial EIA kit (Bachem, San Carlos, Calif.), according to the manufacturer's instructions. All measurements were performed in duplicate and the data represent the mean of two separate assay results.

Real Time RT-PCR Analysis

Total RNA was isolated from freshly enucleated eyes using Trizol Reagent (Invitrogen, Carlsbad, Calif.) according to manufacturer's instructions. Reverse transcription was performed using Enhanced Avian HS RT-PCR kit (Sigma-Aldrich, Inc., St. Louis, Mo.) following manufacturer's instructions. Real time PCR was carried out on real time thermal cycler (iCycler, Bio-Rad Life Sciences, Hercules, Calif.) using iQTM Sybr Green Supermix (Bio-Rad Life Sciences, Hercules, Calif.). The threshold cycle number (Ct) for real-time PCR was set by the cycler software. Optimal primer concentration for PCR was determined separately for each primer pair. Each reaction was run in duplicate or in triplicate, and reaction tubes with target primers and those with Actin primers were always included in the same PCR run. To test the primer efficiencies, the one-step reverse-transcriptase-PCR was run with each target primer. Relative quantification was achieved by the comparative 2^(−ΔΔCt) method 1. The relative increase/decrease of mRNA target X in the experimental group (EG) was calculated using the control group as the calibrator: 2^(−ΔΔCt), where ΔΔCt is {Ct.x [EG]−Ct. Actin [EG]}−{Ct.x[control]−Ct. Actin [control]}. Primer sequences used in this study are shown in Table 1. All the reactions were repeated at least twice.

TABLE 1 Primers used for Real-Time RT-PCR analysis Accession Gene name number Sequences Interleukin-6 NM_031168.1 Forward: 5′-TCGGCAAACCTAGTGCGTTA -3′ (4)* Reverse: 5′-CCAAGAAACCATCTGGCTAGG-3′ (5) IL-1β NM_008361.3 Forward: 5′-AAAGCCTCGTGCTGTCGGACC-3′ (6) Reverse: 5′-CAGCTGCAGGGTGGGTGTGC -3′ (7) TNF-α NM_013693.2 Forward: 5′-AGGCGCCACATCTCCCTCCA-3′ (8) Reverse: 5′-CGGTGTGGGTGAGGAGCACG-3′ (9) ICAM-1 NM_010493 Forward: 5′-AGATGACCTGCAGACGGAAG-3′ (10) Reverse: 5′-GGCTGAGGGTAAATGCTGTC-3′ (11) MCP-1 NM_011333 Forward: 5′-CCCCACTCACCTGCTGCTACT-3′ (12) Reverse: 5′-GGCATCACAGTCCGAGTCACA-3′ (13) β-Actin X03672 Forward: 5′-AGCAGATGTGGATCAGCAAG-3′ (14) Reverse: 5′-ACAGAAGCAATGCTGTCACC-3′ (15) MAS receptor NM_008552 Forward: 5′-AGGGTGACTGACTGAGTTTGG-3′ (16) Reverse: 5′-GAAGGTAAGAGGACAGGAGC-3′ (17) AT1Ra NM_177322 Forward: 5′-ATCGGACTAAATGGCTCACG-3′ (18) Reverse: 5′-ACGTGGGTCTCCATTGCTAA-3′ (19) AT1Rb AK087228 Forward: 5′-AGTGGAGTGAGAGGGTTCAA-3′ (20) Reverse: 5′-GGGCATTGAAGACATGGTAT-3′ (21) *Numbers in parentheses are SEQ ID NOS. Fundus Imaging and Assessment of EAU

Fundus assessment of EAU was performed at day 14 after EAU induction. The pupils were dilated using atropine sulfate and phenylephrine hydrochloride. The mice were anesthetized by intraperitoneal injection of ketamine (75 mg/kg) and xylazine (5 mg/kg) mixture, and Gonak Hypromellose demulcent ophthalmic solution (Akorn, Inc., Buffalo Grove, Ill.) was used on ocular surface. The fundus was imaged using the Micron II small animal retinal imaging AD camera (Phoenix Research Laboratories, Pleasanton, Calif.). Eyes were examined for vasculitis, focal lesions, linear lesions, retinal hemorrhages and retinal detachment. Clinical EAU scoring was performed on a scale of 0-4, as described in detail previously [31]. EAU clinical data shown was representative of two sets of experiments.

Spectral Domain Optical Coherence Tomography (SD-OCT) Imaging and Assessment of EAU Mice

Mice were anesthetized and the pupil dilated as described above. Artificial tears (Systane Ultra, Alcon, Fort Worth, Tex.) were used throughout the procedure to maintain corneal moisture and clarity. SD-OCT images were obtained in mice on 14th day after immunization using the Bioptigen Spectral Domain Ophthalmic Imaging System (Bioptigen, Inc., Durham, N.C.). Images acquired by the software provided from the company. The average single B scan and volume scans were obtained with images centered on optic nerve head. The retinal thickness was measured from five frames of the volume of OCT images and averaged from the intensity peak of boundary corresponding to the vitreo-retinal interface to the intensity peak corresponding to the retinal pigmented epithelium [53]. EAU clinical data shown was representative of two sets of experiments.

Statistical Analysis

Data are expressed as the mean+SD of at least two independent experiments. Differences between mean values of multiple groups were analyzed by one-way analysis of variance with Dunnett's test for post hoc comparisons. A p-value less than 0.05 was considered statistically significant.

Results

Hyperactivity of the renin-angiotensin system (RAS) resulting in elevated Angiotensin II (Ang II) contributes to all stages of inflammatory responses including ocular inflammation. The discovery of angiotensin-converting enzyme 2 (ACE2) has established a protective axis of RAS involving ACE2/Ang-(1-7)/Mas that counteracts the proinflammatory and hypertrophic effects of the deleterious ACE/AngII/AT1R axis. In the present example, we demonstrate that enhancing the systemic and local activity of the protective axis of the RAS by oral delivery of ACE2 and Ang-(1-7) bioencapsulated in plant cells confers protection against ocular inflammation. Both ACE2 and Ang-(1-7), fused with the non-toxic cholera toxin subunit B (CTB) were expressed in plant chloroplasts as described in Example I. In the present example, we show that increased levels of ACE2 and Ang-(1-7) were observed in circulation and retina after oral administration of CTB-ACE2/Ang-(1-7) expressing plant cells. Oral feeding of mice with bioencapsulated ACE2 or Ang-(1-7) significantly reduced endotoxin-induced uveitis (EIU) in mice. Treatment with bioencapsulated ACE2 or Ang-(1-7) also dramatically decreased cellular infiltration, retinal vasculitis, damage and folding in experimental autoimmune uveoretinitis (EAU). Thus, enhancing the protective axis of RAS by oral delivery of ACE2/Ang-(1-7) bioencapsulated in plant cells provide an innovative, highly efficient and cost-effective therapeutic strategy for ocular inflammation such as EIU and EAU.

The ability to deliver drugs efficiently to the retina or the brain remains a key challenge due to anatomic barriers and physiological clearance mechanisms [13]. The Plant chloroplast genetic engineering system to express therapeutic proteins is emerging as a highly efficient, cost-effective approach for therapeutic interventions of many pathologic conditions [12, 14]. In contrast to current protein production systems (mammalian, yeast, or bacteria), the transplastomic system requires no complex production/purification steps [14]. Current biopharmaceuticals are not affordable to more than half of the global population because of use of prohibitively expensive production, purification and delivery systems [14]. However, chloroplasts produce the same biopharmaceuticals at a significantly lower cost by eliminating fermentation, purification, cold chain and sterile delivery systems. Such cGMP facilities to produce plants for human clinical studies are already in use in the US (e.g. Fraunhofer, Delaware, Kentucky Bioprocessing, etc.). Ultimately, the therapeutic proteins will be provided to patients as capsules after lyophilization of plant cells, facilitating prolonged storage at room temperature. In addition, bioencapsulation of therapeutic proteins within plant cell walls enable oral delivery by their protection in the digestive system [14,15]. The bioencapsulated proteins that pass through the stomach are released in the intestine with the aid of commensal bacteria [16,17]. Bacteria inhabiting the human gut have evolved to utilize complex carbohydrates in plant cell wall and are capable of utilizing almost all of plant glycans. So the gut microbes recognize, import, and digest plant cell wall consisting of cellulose, hemicellulose, and pectin. Up to 10% daily energy is obtained from polysaccharide fiber via the symbiotic bacteria living in human gut. These polysaccharides are broken down to sugars and fermented to short fatty acids then absorbed by human gut. Therapeutic proteins enter circulation by receptor monosialotetrahexosylganglioside (GM1) mediated delivery when fused with the non-toxic subunit B of cholera toxin (CTB) as the transmucosal carrier [14, 18-22]. The use of CTB as a transmucosal carrier can facilitate the transportation of conjugated proteins into circulation through its strong binding to GM1 because large mucosal area of human intestine (approximately 1.8-2.7 m2 [23] facilitates CTB to bind an intestinal epithelium cell up to a maximum of 15,000 CTB [24] and the rapid turnover of cell-associated GM1 receptor on the cell [25]. Furthermore, the GM1 gangliosides receptors are also found in the plasma membranes of most cells, particularly most abundant in the nervous system and retina [26], allowing efficient uptake of CTB fusion protein in the brain and retinal cells as observed in our recent study [12]. Considering the proven anti-inflammatory actions of ACE2 and Ang-(1-7) and the ability of CTB to cross the epithelial barrier and facilitate neuronal uptake, we hypothesized that oral delivery of ACE2 and Ang-(1-7) fused with CTB bioencapsulated in plant cells will enhance both systemic and local activity of the protective axis of RAS and confer protection against ocular inflammation. In this example, we tested this hypothesis in two mouse models of ocular inflammation. We observed that oral administration of CTB-ACE2/Ang-(1-7) bioencapsulated in plant cells reduced ocular inflammation in both EIU and EAU models, providing proof-of-concept that enhancing the protective axis of RAS by oral delivery of ACE2/Ang-(1-7) bioencapsulated in plant cells provides an innovative, highly efficient and cost-effective therapeutic strategy for ocular inflammation such as uveitis and autoimmune uveoretinitis.

Results

Creation of Transplastomic Plants Expressing CTB-ACE2/-Ang-(1-7)

The CTB-fused therapeutic genes were cloned into the chloroplast transformation vector, pLD. The hinge site (FIG. 1) was introduced between CTB and therapeutic proteins to avoid steric hindrance and facilitate formation of pentameric structure of CTB fused to therapeutic proteins, when expressed in chloroplasts. Pentameric structure of CTB plays a critical role in translocating fusion proteins into epithelial cells via the GM1 receptor. Furin cleavage site (FIG. 1a ) facilitates release of therapeutic proteins from CTB after transmucosal delivery. The furin protease is ubiquitously present in all cell and tissue types and the consensus cleavage site is well characterized [27]. The introduction of the consensus furin cleavage site between CTB and fused proteins will ensure efficient release of the therapeutic proteins from CTB into the circulation. For the site-specific integration of the CTB-ACE2/-Ang-(1-7) expression cassette into chloroplast genome, the cassette was flanked by trnI and trnA sequence, which are homologous to endogenous chloroplast sequence. Light regulated strong chloroplast promoter, PsbA, was used to express the fusion gene. To screen chloroplast transformants, aminoglycoside 3′-adenylyltransferase gene (aadA) was driven under the control of ribosomal rRNA promoter (Prrn) to disarm the inhibitory action of spectinomycin on chloroplast translation (FIG. 1). The sequence-confirmed chloroplast transformation vectors were bombarded onto leaves to create the transplastomic plants expressing CTB-ACE2 and CTB-Ang-(1-7), using biolistic particle delivery system. Shoots emerged from spectinomycin containing regeneration medium were investigated for the site specific integration of the expression cassette into the chloroplast genome, using PCR analysis. The specific primer sets were designed to amplify fragments in the size of ˜1.65 kb with 3P/3M for both CTB-ACE2 and CTB-Ang-(1-7), ˜4.5 and ˜2.2 kb with 5P/2M for CTB-ACE2 and CTB-Ang-(1-7), respectively, and ˜3.0 and ˜1.1 kb with 5P/R for CTB-ACE2 and CTB-Ang-(1-7), respectively. Positive shoots displaying the expected right size fragments were subjected to two more rounds of tissue culture under antibiotic selection to achieve homoplasmy. The homoplasmic plants were confirmed by Southern blot analysis (data not shown), transferred and grown in a temperature- and humidity-controlled greenhouse. The expression level of CTB-fused therapeutic proteins of mature leaves was measured quantitatively using densitometry and Image J or ELISA with known amount of CTB proteins to generate the standard curve. The expression level was up to 2.14% and 8.7% of total leaf protein for CTB-ACE2 and CTB-Ang-(1-7) at their peak, respectively (data not shown). For consistency of batches between harvests, leaves were always harvested at 6 pm to maximize accumulation of therapeutic proteins expressed under the control of a light regulated promoter (psbA). Also, only mature leaves were chosen for harvest to maintain similar expression levels of the proteins between batches. While it is difficult to precisely control expression levels at the time of harvest, dosage is precisely determined after lyophilization and the same dose is delivered by varying the weight of lyophilized powder in each capsule or gavage.

Characterization of CTB-Fused Therapeutic Proteins Expressed in Chloroplasts

CTB was used as a carrier to allow therapeutic proteins to pass through both epithelial and blood-retinal barrier [12], which is mediated by the interaction between pentameric CTB and GM1 receptor. To investigate the proper folding and assembly of the pentameric structure of CTB-fused therapeutic proteins in chloroplasts, western blot analysis was performed with CTB-Ang-(1-7). Proteins were extracted under non-denaturing conditions, followed by either treatment with or without denaturing agents, prior to separation on SDS-acrylamide gel. There was negligible change in polypeptide profile oligomeric structures of CTB-Ang-(1-7) when protein was treated with DTT alone (FIG. 12a ). In contrast, boiling samples showed dramatic change in polypeptide patterns (FIG. 12a ). This is consistent with the previous studies on dissociation of CTB pentameric structure [28]. Multiple interactions between CTB monomers, such as 30 hydrogen bonds, 7 salt bridges and hydrophobic interactions, make pentameric structure of CTB highly resistant to the dissociation so the pentamer structure is not affected by denaturants such as SDS and DTT. However, the structure can be dissociated by using heat energy. This could be due to the difference in accessibility of denaturing agents to their targets. The access of DTT to the intramolecular disulfide bond of monomer is not easy unless pentameric structure dissociates first, due to the intimate interactions between monomers described above. As expected, both denaturing agents showed no high molecular weight oligomers (pentamer-pentmer interactions), but dimeric and monomeric forms of CTB-Ang-(1-7) were observed (FIG. 12a ). The intramolecular disulfide bond of CTB monomer was easily disrupted by DTT after boiling than either boiling alone or DTT alone (FIG. 12a ). Boiling allowed easy access of DTT to the internal disulfide bond by breaking intimate interactions between CTB monomers (hydrogen bonds and salt bridges) (FIG. 12a ). From these results, it is evident that the disulfide bond of CTB-Ang-(1-7) monomer was properly formed and the interactions between the monomers of pentameric structure of CTB-Ang-(1-7) were well established in chloroplasts.

For clinical application, long-shelf life and stability of therapeutic protein expressed in plants are very important for successful and cost-effective treatment. Therefore, lyophilized CTB-fused therapeutic protein leaves were fully characterized. The weight of lyophilized leaf is usually reduced by 90% to 95% as a result of removal of water from plant cells, leading to more total protein per mg of leaf powder. Extraction of concentrated proteins from lyophilized leaf materials needs more volume of the extraction buffer because the amount of water lost in the process of lyophilization is slowly reabsorbed by the dried materials. For quantitation of lyophilized leaf materials, 10 mg of lyophilized powered leaf materials were resuspended in 300 μl extraction buffer in contrast to 100 mg of fresh leaf materials in the same volume of extraction buffer. Then the extracted total proteins were used for quantification for comparison between fresh and lyophilized leaf materials. Western blot analysis of the CTB-Ang-(1-7) showed that the band patterns between fresh and 3-month old lyophilized leaf materials were identical (FIG. 12b ), confirming stability during lyophilization and prolonged storage at room temperature. As seen in the blot, twenty-time less lyophilized protein sample loaded showed similar band intensities to fresh leaf proteins (FIG. 12b ). The quantity of CTB-Ang1-7 in fresh and lyophilized leaves was measured using immunoblots (FIG. 12b ) and Image J software; the quantity of CTB-Ang-(1-7) increased 14.3 times in lyophilized leaves when compared to fresh leaves (FIG. 12c ). The lyophilized CTB-ACE2 leaves showed 20.5 fold more CTB-Ace2 than fresh leaves when quantified using ELISA (FIG. 12d ).

In this study, we observed that there was no damage or loss of the fusion protein (FIG. 12b ) under the optimized lyophilization conditions. Moreover, the intactness of the pentameric structure of the lyophilized CTB-fused proteins was well preserved up to 15 months at room temperature, as confirmed in GM1 binding assay which showed binding affinity of the lyophilized CTB-fused proteins to GM1 as compared to respective positive control, CTB (FIG. 12e ). Taken together, the homoplasmic transplastomic plants expressing CTB-ACE2 and -Ang-(1-7) were created and the fusion protein was properly expressed, folded, and assembled in chloroplasts. The folding, assembly and functionality of therapeutic proteins were well preserved in lyophilized leaves.

ACE2 activity assay using protein extracts isolated from plant leaves showed that plant cell expressed human ACE2 is enzymatically active (FIG. 13a ). To investigate the in vivo potential of CTB-ACE2 and CTB-Ang-(1-7) to cross the intestinal barrier and tissue uptake, wild type C57B1/6J mice were fed with either fresh (F, ˜500 mg/mouse), or ten-fold less lyophilized (L, ˜50 mg/mouse) CTB-ACE2, or control untransformed (WT) leaf materials for three days, mice were sacrificed at 5 hr after the last gavage. Circulatory and retinal ACE2 and Ang-(1-7) levels were measured by ACE2 activity assay, EIA and Western blotting (FIG. 13). ACE2 protein can be detected in both serum and retina 5 hours after oral gavage (FIG. 13b ). Oral administration of either fresh frozen (F) or lyophilized (L) CTB-ACE2 transgenic leaf materials resulted in an increase of approximately 40% and >20% in ACE2 enzymatic activity in serum and retina, respectively when compared to WT leaf fed mice (FIG. 13c ). There was a 15% increase in plasma and nearly 50% increase in Ang-(1-7) peptide level in the retina in CTB-Ang-(1-7) expressed leaf material fed group, detected by Ang-(1-7) specific EIA kit (FIG. 13d ).

Oral Administration of Bioencapsulated CTB-ACE2 and CTB-Ang-(1-7) Reduced the Infiltration of Inflammatory Cells Induced by EIU

We next examined the effects of ACE2 and Ang-(1-7) on endotoxin-induced infiltration of inflammatory cells such as leucocytes and monocytes in the iris, ciliary body, anterior chamber, and posterior chamber of the eye. Sagittal sections were stained with H&E and examined under bright field microscope. The histological evaluation of LPS injected eyes from mice fed with WT leaf revealed severe signs of uveitis with massive infiltration of inflammatory cells into the iris and ciliary body (ICB) (140±21 cells/section), anterior chamber (265±52 cells/section) and the posterior chamber (202±37 cells/section) (FIG. 14). Prophylactic treatment with CTB-ACE2 showed significantly diminished uveitis and reduced number of inflammatory cells into the ICB (60±09 cells/section), anterior chamber (96±15 cells/section) and also into the posterior chamber (82±15 cells/section). Similar results were observed in ICB (70±15 cells/section), anterior chamber (114±36 cells/section) and the posterior chamber (28±15 cells/section) when animals pretreated with CTB-Ang-(1-7) expressed leaf material (FIG. 14).

The therapeutic effect of different doses of CTB-ACE2 in EIU was evaluated using lyophilized leaf materials. Oral feeding of lyophilized CTB-ACE2 at 50 mg/day significantly prevented inflammatory cell infiltration into the iris and ciliary body, anterior chamber and posterior chamber to the same extent as the fresh leaf material at 500 mg/day; CTB-ACE2 feeding at 25 mg/day had moderate but significant protection, whereas 12.5 mg/day did not show any protective effect in EIU (FIG. 14C).

Oral Administration of Bioencapsulated CTB-ACE2 and CTB-Ang-(1-7) Reduced the Expression of the Inflammatory Cytokines in EIU Eyes

To investigate the effects of ACE2 and Ang-(1-7) on the expression of inflammatory cytokines in EIU eyes, the mRNA levels of cytokine genes were determined by real-time RT-PCR. In the WT leaf fed mice, LPS caused a significant increase in mRNA levels of Interleukin-6 (IL-6), Interleukin-1β (IL-1β), Tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) and this increase was reduced in mice fed with ACE2 or Ang-(1-7) leaf materials (FIG. 15a ). These results suggest that ACE2 and Ang-(1-7) reduced infiltration of inflammatory cells and cytokine production through suppressing their gene expressions during EIU. To investigate the molecular mechanisms of leucocyte recruitment, the mRNA levels of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein (MCP-1) were measured in EIU eyes. The expression of both ICAM-1 and MCP-1 was significantly increased in LPS induced EIU eyes in mice treated with WT leaf material and was significantly reduced in mice fed with CTB-ACE2 or CTB-Ang-(1-7) (FIG. 15a ).

The Impact of Oral Administration of Bioencapsulated CTB-ACE2 and CTB-Ang-(1-7) on the Expression of the Retinal RAS Genes During EIU

In addition to circulating RAS, all components of RAS have been detected in the retina and a local retina RAS may play an important role in modulating local immune responses [10,11,29]. We compared ocular mRNA levels of the key RAS genes in animals fed with ACE2 and Ang-(1-7) expressing leaf materials as well as untransformed WT leaf materials. LPS-induced EIU resulted in increased expression of both ACE and ACE2, however prominent increase in ACE (more than 4-fold increase) than ACE2 (less than 2-fold increase), resulted in increased ratio of ACE/ACE2 (FIG. 15b ). CTB-ACE2 or CTB-Ang-(1-7) oral feeding normalized the shift of ACE/ACE2 ratio (FIG. 15b ). The mRNA levels for receptors for Ang II (AT1Ra, AT1Rb) were also increased in EIU mice fed with control leaf material (˜4-fold and 1.7-fold respectively) (FIG. 15b ), both of which were significantly decreased in mice fed with CTB-ACE2 or CTB-Ang (1-7). There was a slight but significant increase in Mas, the receptor for Ang-(1-7). Interestingly the Mas mRNA level in the retina was further increased in mice fed with CTB-ACE2 (˜2-fold increase), and even more increase in mice fed with CTB-Ang-(1-7) leaf material (˜3-fold increase) (FIG. 15b ), suggesting a possible feed-forward regulatory response in local retinal RAS.

Oral Delivery of Bioencapsulated CTB-ACE2 and CTB-Ang-(1-7) Attenuated Autoantigen Induced Uveoretinitis

Experimental autoimmune uveoretinitis was induced in an autoimmune susceptible B10.RIII mouse strain by active immunization using a peptide derived from the retinal protein IRBP [30]. Evident inflammatory reactions such as mild to severe vasculitis, focal lesions, large confluent lesions, retinal hemorrhages and folding, corneal edema etc., were observed in WT leaf fed animals by fundoscopy examination at day 14 of EAU induction (FIG. 16A, a-b). However, mice fed with CTB-ACE2 or -Ang-(1-7) leaf materials showed significantly reduced inflammatory reactions (FIG. 16A, c-d, and e-f). The clinical scoring, using the criteria reported by Copland et al [31], showed that eyes from CTB-ACE2 or CTB-Ang-(1-7) fed animals had significantly improved clinical scores (EAU grade, 2.3±1.2 and 2.3±1.3 respectively) compared to eyes from animals fed with WT leaf (EAU grade, 3.4±0.53) (FIG. 16B).

The uveoretinitis was also evaluated by OCT imaging on day 14 after immunization with IRBP. In few cases severe retinal pathology such as high level of cellular infiltration, edema, folds, and hemorrhages limited OCT resolution of retinal layers. In most cases, intravitreal cellular infiltration, retinal vasculitis, disorganized retinal layers and increased retinal thickness due to retinal folds and edema, can be easily visualized with OCT imaging as shown in FIG. 17a in untreated or WT leaf material treated animals, these pathologies are much improved in mice treated with CTB-ACE2 or Ang-(1-7) (FIG. 17a ). Treatment with CTB-ACE2 or CTB-Ang-(1-7) leaf materials significantly reduced EAU-induced increased retinal thickness (269±32 μm and 241±52 μm respectively) compared to eyes treated with WT leaf (316±32 μm) (FIG. 17b ).

Oral Administration of CTB-ACE2 and CTB-Ang-(1-7) Ameliorates Histological Findings in the EAU Mice

Histological examination on day 14 showed a severe intraocular inflammation evidenced by massive infiltration of inflammatory cells, intensive retinal vasculitits, and changes in the retinal thickness, folding of retina, as well as photoreceptor damage in the WT leaf fed mice (FIG. 18a ). However, only scattered inflammation of inflammatory cells and minor retinal folding was observed in CTB-ACE2 or CTB-Ang-(1-7) treated animals (FIG. 18a ). Histopathological grading, using the criteria reported by Thurau et al. [32] showed that WT leaf fed eyes (EAU grade, 2.95±0.717) had significantly severe inflammation as compared to CTB-ACE2 (EAU grade, 1.1±0.616) and CTB-Ang-(1-7) (EAU grade, 0.92±0.535) expressed leaf fed eyes (FIG. 18b ). Similarly significantly higher numbers of inflammatory cells were observed in the posterior chamber of WT leaf fed mice compared to the CTB-ACE2/Ang-(1-7) expressed leaf fed mice (FIG. 18b ).

To determine whether CTB-Ang-(1-7) treatment can also improve EAU after its onset or during its progression, daily oral feeding was delayed to day 5 and day 10 after EAU induction, and continued to day 14 when mice were euthanized for evaluation. We observed that feeding from day 5 onward up to day 14 after IRBP injection is equally as effective as feeding from day 0, but feeding started at 10 after EAU induction had no improvement of ocular pathology (FIG. 19).

Discussion

In this study, we have developed an expression system to generate high levels of human ACE2 and Ang-(1-7) within plant chloroplasts using transplastomic technology. Oral gavage of plant cells expressing ACE2 and Ang-(1-7) fused with CTB in mice resulted in increased circulating and retinal levels of ACE2 and Ang-(1-7), reduced ocular inflammation in two different models: endotoxin-induced uveitis (EIU) and autoantigen induced experimental autoimmune uveoretinitis (EAU).

Among many advantages of transplastomic technology, the high copy number of a transgene, up to >10,000 copies per cell, is a key to successful high level expression of therapeutic proteins in chloroplasts. However, this advantage could be limited when human transgenes are not codon-optimized because the preference of codon usage of chloroplast is different from that of eukaryotic cell. The codon adjustment for chloroplast expression system is crucial for the efficient expression of human genes [33]. So, the relatively low expression ACE2 over the Ang-(1-7) is probably due to the use of native human gene sequence (805 amino acids). However, several other tools were incorporated into our system to offset low expression of the transgenes so that the contents of therapeutic proteins expressed in chloroplasts can be increased. For example, the expression of therapeutic proteins under the control of light-regulated strong chloroplast promoter, harvest of mature leaves at the end of day, and lyophilization of the harvested leaves. The chloroplast psbA promoter is light regulated and therefore harvesting leaves before sunset maximizes accumulation therapeutic proteins.

Moreover, the amount of therapeutic proteins in plant leaves can be concentrated by lyophilization (FIGS. 12c and 12d ). The process of dehydration of the leaves under vacuum at −51° C. for 3 days can significantly reduce the risk of microbial contamination [14]. The long-term shelf life of the lyophilized proteins at room temperature can also decrease the cost associated with the cold chain of current injectable proteins [14]. The effect of lyophilization on the stability of proteins expressed in chloroplasts has been extensively studied in our lab under the condition at which temperature and pressure were set up at −51° C. and 27 mTorr, respectively, according to the chart of vapor pressure of ice provided by manufacturer. When the effect of duration of lyophilization was investigated, large protective antigen (PA, 83 kDa) expressed in lettuce chloroplasts was stable at 24, 48, and 72 hrs of lyophilization. In addition, the lyophilized antigen was more stable at room temperature than the commercially purified antigens stored at low temperatures. Further, the lyophilized PA was found to be stable up to 15 months at room temperature without any degradation [14]. Other transplastomic plants expressing CTB-exendin 4 [21] and CTB-Factor VIII [34] showed similar stability of fusion proteins and protection of their assembly, folding and disulfide bonds similar to fresh leaves.

Drug delivery to different compartments of the eye, particularly to the posterior segment of the eye, is a major challenge due to several barriers formed by both anatomical structure and the protective physiological mechanisms of the eye[13]. Large molecular weight therapeutics such as peptides/proteins and oligonucleotides are delivered mostly via intravitreal route. However, frequent administration via this route is often associated with many complications such as retinal detachment, endophthalmitis, and increased intraocular pressure [35, 36]. We demonstrate that oral administration of CTB-ACE2 increased ACE2 activity in sera and retina. Similarly increased level of plasma and retinal Ang-(1-7) was observed when the animals were orally administered with CTB-Ang-(1-7), as observed in previous study of oral delivery of bioencapsulated proteins across blood-brain and retinal barriers [12]. The increased level of the Ang-(1-7) could be stemmed from the fusion with CTB. The short peptide of Ang-(1-7) fused to CTB becomes stabilized in a form of pentameric structure in plant cells (FIG. 12a ). However, only monomers are observed in sera once delivered into circulation. Considering that the efficiency of furin cleavage site depends on the flanking amino acid sequence of the fused protein [37], Ang-(1-7) fusion to CTB did not offer optimal furin cleavage site. Thus, the cleavage between CTB and Ang-(1-7) is not likely to be fast or efficient once the fusion protein gets into sera. In addition, the CTB fusion provides N-terminal protection for Ang-(1-7) so its stability is extended for several hours, while injectable Ang-(1-7) has a very short half-life in sera [38, 39]. Although the Ang-(1-7) level was increased in both plasma and retina (FIG. 13d ), the level of Ang-(1-7) increase in plasma was less than in retina (FIG. 13d ). This difference could be attributed to the increased retention in tissues (cells) and to their rapid clearance in the sera by proteases. Similar result was also observed in our previously published study in which GFP level was shown several fold higher in tissues than in sera [12, 19]. Although EIU was originally used as a model of anterior uveitis because of its characteristic infiltration of leucocytes into the anterior chamber of the eye [4], growing evidences suggest that it also involves inflammation in the posterior segment of the eye, with recruitment of leucocytes that adhere to the retinal vasculature and infiltrate the vitreous cavity [3]. Our results demonstrate that enhanced level of ACE2/Ang-(1-7) in both circulation and ocular tissues suppressed the endotoxin-induced ocular inflammation, which is evident from significantly reduced number of infiltrating inflammatory cells in the iris-ciliary body, anterior and posterior chambers. This result is consistent with studies showing anti-inflammatory property of ACE2/Ang-(1-7) in other disease models [9]. The dose dependent study using bio-encapsulated lyophilized CTB-ACE2 in EIU model further confirmed that a dose of ˜50 mg/day for four days can significantly prevent the endotoxin-induced inflammation. We also showed that increased ACE2/Ang-(1-7) significantly suppressed the LPS-induced ocular expression of IL-6, IL-1β, TNF-α and VEGF. It has been reported that in EIU model, leucocytes are markedly attracted to inflamed ocular tissues such as the iris [40], vitreous cavity [41] and retina [42], with neutrophils and macrophages being major leucocyte constituents. MCP-1 is known as one of the important factors for leucocyte recruitment, and is up-regulated during EIU [43] whereas ICAM-1 is known to be the key molecule of leucocyte adhesion and/or transmigration [44]. Our study demonstrates that mice fed with ACE2/Ang-(1-7) leaf materials showed decreased ocular expression of MCP-1 and ICAM-1 in EIU eyes, contributing to the diminished inflammatory response by inhibiting leucocyte recruitment and adhesion in the ocular tissue. These results are consistent with the histopathology observation that LPS-induced acute inflammation caused the increase of inflammatory cell recruitment, while ACE2/Ang-(1-7) treatment significantly reduced the inflammatory cells in iris/ciliary body, anterior and posterior chamber.

It has been shown that ACE2/Ang-(1-7) may directly reduce inflammatory responses in immune cells such as macrophages [45]. Our study also shows modulatory ability of ACE2/Ang-(1-7) on local immune response and cytokine/chemokine expression. In fact the Mas receptor is expressed not only in retinal vascular cells, astrocytes and muller glia, but also in retinal neurons, consistent with its role in neuro-vascular and immune response modulation. Moreover, our results show that eyes with EIU are associated with decreased expression of ACE2 and Mas receptor and increased expression of vasoconstrictive axis genes such as ACE, AT1Ra, AT1Rb during EIU. This is prevented by ACE2/Ang(1-7), suggesting that the anti-inflammatory effect of ACE2/Ang-(1-7) may be associated with Mas receptor and ACE2 up-regulation, and down-regulation of ACE and AT1Ra/AT1Rb, resulting in reduction of ocular inflammation.

In this study, we also investigated the effect of oral administration of CTB-ACE2/Ang-(1-7) on the development of EAU in mice and showed significant improvement of EAU eyes in mice treated with CTB-ACE2/Ang-(1-7). The pathogenesis of EAU is different from EIU. EAU is defined primarily as posterior segment disease as the target antigens reside in the retina and characterized by cellular infiltrates, retinal folds, detachment, granulomatous infiltrates in the retina and choroid, vasculitis, retinal neovascularization, mild to severe photoreceptor loss [32]. Histopathological examination confirmed a significant overall reduction of disease severity in the CTB-ACE2/Ang-(1-7) treatment groups evaluated by non-invasive funduscopy and OCT imaging methods. Furthermore, the retinal detachment, photoreceptor layer damage, infiltration of inflammatory cells was markedly prevented by CTB-ACE2/Ang-(1-7) treatments. Some of the fundoscopically normal-looking eyes showed few foci of very mild cellular infiltrates on histological evaluation. This is consistent with the findings from OCT imaging, demonstrating a better correlation of histological findings and pathological changes revealed by non-invasive OCT imaging in the retina during EAU. Thus, oral administration of CTB-ACE2/Ang-(1-7) from the induction to peak of EAU was able to ameliorate the progression of disease evaluated by clinical funduscopic score, OCT imaging and histopathological observation. Moreover, delayed oral administration of CTB-ACE2/Ang-(1-7) from day 5 after EAU induction was also able to decrease the progression of EAU.

Increasing evidence has shown that shifting the balance of RAS towards the protective axis by activation of ACE2 or its product, Ang-(1-7) is beneficial and anti-inflammatory [9,46]. Our findings also demonstrate that oral administration of CTB-ACE2/Ang-(1-7) provides robust protective anti-inflammatory effects against the pathophysiology in both EIU and EAU models.

In conclusion, this study provides proof-of concept for production of therapeutically active ACE2/Ang-(1-7) bioencapsulated in plant cells for cost effective oral therapy for ocular applications and enhancing ACE2/Ang-(1-7) using this approach may provide a new avenue and a novel therapeutic strategy for the treatment of acute uveitis, autoimmune uveoretinitis and other ocular diseases.

REFERENCES FOR EXAMPLE 2

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Example 3 Codon Optimization of ACE2 and Ang-(1-7)

In a recent study, Nakamura et al. disclose the importance of compatibility between the psbA 5′-UTR and its 5′ coding sequence when using codon-optimized heterologous genes. Previously reported codon optimization studies used only smaller eukaryotic coding sequences (<30 kDa). However, there is a great need to express larger human genes (eg. Human blood clotting factor VIII >200 kDa) that would require optimization of not only codons but also compatibility with regulatory sequences for optimal translation initiation, elongation and greater understanding of tRNAs encoded by the chloroplast genome or imported from the cytosol. However, no systematic study has been done to utilize the extensive knowledge gathered by sequencing several hundred chloroplast genomes to understand codon usage and frequency of highly expressed chloroplast genes.

Among the 140 transgenes expressed in chloroplasts, >75% use the psbA regulatory sequences (Daniell et al., 2016). Most importantly, compatibility between the 5′-UTR of psbA and its coding region is important for efficient translation initiation (Nakamura et al., 2016). For these reasons, a new codon optimization program was developed using codon usage of the psbA genes from 133 sequenced chloroplast genomes (FIG. 20A). We first investigated expression of synthetic genes using only the most highly preferred codon for each amino acid, which is referred to as the “old” version in this study. When this resulted in even lower levels of expression than the native gene (see data presented below), a “new” codon optimizer algorithm was developed using codon usage hierarchy observed among sequenced psbA genes. See FIGS. 20B, C and D. Therefore, most of the rare codons in heterologous genes were modified based on codons with >5% frequency of use in the psbA genes.

In this study, the CNTB coding sequence was not codon-optimized because of its prokaryotic origin and high AT content (65.37%). Most importantly, the expression level of CNTB (native sequence) fused with proinsulin in tobacco chloroplasts reached up to 72% of total leaf protein (Ruhlman et al., 2010) and 53% of total leaf protein in lettuce chloroplasts (Boyhan and Daniell, 2011), indicating that there is no limitation on translation of the CNTB coding sequence in chloroplasts.

When the psbA-based codon table is compared with total chloroplast codon usage tables, which are generated based on all chloroplast genes of Lactuca sativa (57,528 codons from 189 coding sequences) or Nicotiana tabacum (34,756 codons from 137 coding sequences) (Nakamura et al., 2000), there was no significant difference in AT content of coding sequences; it varied between 59.59% and 61.76%. However, there are striking differences between psbA-based and total chloroplast gene-based codon tables when individual codons are compared. Ten rare codons identified in the psbA codon table (<5% usage frequency), (which were not used in codon-optimized sequences in the new version) were not identified as rare codons in the total chloroplast gene codon usage table. For example, CTC (leucine) is almost never used in psbA genes (0.1%) but this codon's usage is >6.4% in the lettuce or 7.5% in the tobacco total chloroplast codon table. Likewise, CGG (arginine) and ACG (threonine) are used with frequencies of 0.5% and 0.8%, respectively, in the psbA-based codon table, but the same codons are used with 7.6% and 9.6% frequency in lettuce or 8.3% and 10.8% frequency in tobacco total codon tables. Inadequate identification of rare codons in total chloroplast gene codon tables could be due to the averaging effect of combining highly and poorly expressed genes regardless of their translation efficiency in chloroplasts. This is true for the hierarchy in codon usage among synonymous codons. For example, the 5th codon in the hierarchy for leucine is CTG, which is used at a frequency of 3.7% in the psbA-based codon table; the difference in usage between the 6th codon (CTC, 0.1%) is 37 fold. However, there is no difference in the percentage of codon usage between 5th and 6th codons in lettuce or tobacco chloroplast gene codon tables; the lettuce codon table shows 6.7% (CTG, 5th) and 6.4% (CTC, 6th) frequency and the tobacco table shows 7.5% (5th codon) and 7.1% (6th codon) frequency. The disadvantage of using a total chloroplast gene-based codon table is quite obvious: real differences in codon preference in translation are masked, as reported previously (Surzycki et al., 2009).

Expression of Angiotensin Converting Enzyme in Lettuce Chloroplasts.

Because of low expression level of native ACE2 in transplastomic plants, the creation of a codon optimized ACE2 polypeptide is described in the present example. To improve the production ACE2 in chloroplasts, we first developed new software for codon optimization by creating a database for codon usage of psbA genes from 133 plant species and an algorithm was developed to replace rare codons with preferred codons. The chloroplast psbA gene was chosen as a model because this is the most highly expressed chloroplast gene. The psbA based codon table showed no significant difference in AT content of coding sequences when compared to total chloroplast codon usage tables generated using all lettuce (57,528 codons from 189 coding sequences). However, there are major differences between a single gene based codon table and total gene based codon tables in the usage frequency of individual codons and hierarchy in codon usage. Further, eleven codons identified as rare codons in psbA gene based codon table were not identified as rare codons in chloroplast total gene based codon tables. For example, CTC for leucine is almost not used in psbA genes (0.1%) but the same codon is used at the frequency of 6.4% in lettuce; CGG for arginine in psbA genes (0.5%) but at the frequency of 7.6% in lettuce total chloroplast. Thus, codon-optimized ACE2 were optimized by changing the rare codons and codons usage frequency to resemble the chloroplast psbA gene (FIG. 20A). 481 codons including 59 rare codons out of 805 codons of ACE2 were replaced according to the psbA codon distribution (FIGS. 20B, C, and D). As a result, AT content of the ACE2 gene was increased from 57% to 62.46%, and the frequency of codon usage is similar to psbA gene (FIG. 21A, native, FIG. 21B, optimized). Optimized sequences were then synthesized by GenScript Inc. Genetically modified lettuce lines were created by bombardment of chloroplast vectors into sterile leaves using the gene gun. Transplastomic lettuce shoots confirmed by PCR were cut and subjected to two further rounds of selection. After selection, transplastomic lettuce plants were transferred to greenhouse (FIGS. 22A-22D). Total leaf protein was extracted from native and codon-optimized transplastomic plants and the protein expression level was compared by Western blot analysis. The transplastomic lettuce plants expressing codon-optimized ACE2 showed 7.7-fold higher expression than the native gene (FIG. 23).

CONCLUSION

The present example provides a codon optimized CTB-ACE2 polypeptide sequence which is expressed at significantly higher levels in lettuce chloroplasts. This increased expression should enhance the beneficial cardioprotective effects described in Example I and improve the anti inflammatory effects described in Example 2.

While a number of embodiments have been shown and described herein in the present context, such embodiments are provided by way of example only, and not of limitation. Numerous variations, changes and substitutions will occur to those of skilled in the art without materially departing from the invention herein. For example, the present invention need not be limited to best mode disclosed herein, since other applications can equally benefit from the teachings. Also, in the claims, means-plus-function and step-plus-function clauses are intended to cover the structures and acts, respectively, described herein as performing the recited function and not only structural equivalents or act equivalents, but also equivalent structures or equivalent acts, respectively. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims, in accordance with relevant law as to their interpretation. 

What is claimed is:
 1. A method for the treatment of pulmonary hypertension comprising oral administration of an effective amount of a composition comprising lyophilized plant material comprising i) a fusion protein comprising angiotensin-converting enzyme 2 (ACE-2) and cholera non toxic B subunit (CTB); or ii) a fusion protein comprising angiotensin-(1-7) (Ang-(1-7)), and CTB or i) and ii) in combination to a patient in need thereof, said composition exerting a cardioprotective effect comprising at least one of improved right heart function, decreased pulmonary vessel wall thickness, and a lack of altered basal system blood pressure, said method optionally comprising assessing said effects after administration of said composition.
 2. The method of claim 1, said composition comprising lyophilized plant material obtained from a homoplasmic plant expressing said fusion protein comprising ACE-2 and CTB.
 3. The method of claim 1, said composition comprising lyophilized plant material obtained from a homoplasmic plant expressing said fusion protein comprising Ang-(1-7) and CTB.
 4. The method of claim 3, said composition further comprising lyophilized plant material obtained from a homoplasmic plant expressing said fusion protein comprising ACE-2 and CTB.
 5. The method of claim 2, wherein said homoplasmic plant is lettuce and said fusion protein comprising ACE-2 and CTB is encoded by a sequence comprising SEQ ID NO: 24 or SEQ ID NO:
 26. 6. The method of claim 3, said fusion protein comprising Ang-(1-7) and CTB further comprising a hinge peptide and/or a furin cleavage site between said Ang-(1-7) and said CTB. 